Rna interference mediated inhibition of gene expression using short interfering nucleic acids (sina)

ABSTRACT

The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of gene expression and/or activity, and/or modulate a gene expression pathway. Specifically, the invention relates to double-stranded nucleic acid molecules including small nucleic acid molecules, such as short interfering nucleic acid (siNA) molecules that are capable of mediating or that mediate RNA interference (MAO against target gene expression.

This application is a continuation of U.S. patent application Ser. No.15/947,248, filed Apr. 6, 2018, which is a continuation of U.S. patentapplication Ser. No. 14/984,065, filed Dec. 30, 2015, which is acontinuation of U.S. patent application Ser. No. 13/881,415, filed Oct.25, 2011, which is a National Stage Entry of PCT Application No.PCT/US2011/057663, filed Oct, 25, 2011, which claims the benefit of U.S.Provisional Patent Application No. 61/408,428 filed Oct. 29, 2010 andU.S. Provisional Patent Application No. 61/408,303 filed Oct. 29, 2010,all of which are incorporated herein in their entireties.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 CFR §1.52(e)(5), is incorporated herein by reference. The sequence listingtext file submitted via EFS contains the file “SequenceListing134,”created on Oct. 18, 2011, which is 328,412 bytes in size.

FIELD OF THE INVENTION

The present invention relates to compounds, compositions, and methodsfor the study, diagnosis, and treatment of traits, diseases andconditions that respond to the modulation of gene expression and/oractivity. The present invention is also directed to compounds,compositions, and methods relating to traits, diseases and conditionsthat respond to the modulation of expression and/or activity of genesinvolved in gene expression pathways or other cellular processes thatmediate the maintenance or development of such traits, diseases andconditions. Specifically, the invention relates to chemically modifiedshort interfering nucleic acid (siNA) molecules capable of mediating RNAinterference (RNAi) against gene expression, including cocktails of suchsiNA molecules and formulations of such siNA molecules. Such siNAmolecules and are useful, for example, in providing compositions toprevent, inhibit, or reduce various diseases, traits and conditions thatare associated with gene expression or activity in a subject ororganism.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to RNAi. Thediscussion is provided only for understanding of the invention thatfollows. The summary is not an admission that any of the work describedbelow is prior art to the claimed invention.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Bass,2000, Cell, 101, 235; Fire et al., 1998, Nature, 391, 806; Hamilton etal., 1999, Science, 286, 950-951; Lin et al., 1999, Nature, 402,128-129; Sharp, 1999, genes & Dev., 13:139-141; and Strauss, 1999,Science, 286, 886). The corresponding process in plants (Heifetz et al.,International PCT Publication No. WO 99/61631) is commonly referred toas post-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire et al., 1999, Trendsgenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single-stranded RNA or viral genomicRNA.

The therapeutic potential of RNAi lies in the ability to modulate geneexpression in a sequence specific manner by harnessing a highlyconserved, robust endogenous mechanism of action. This endogenousmechanism of action vastly expands upon the number of available targetsfor disease modification when compared to existing small molecule andbiologic modalities. Nevertheless, a opposed to exogenously suppliedsmall molecule and biologic modalities, the RNA molecules that serve astriggers for RNAi are not well suited for administration due to theirinherent instability, especially in biologic systems. This problem hasbeen addressed through innovation, both in terms of chemicalmodification of RNA triggers (see U.S. Ser. No. 10/444,853, published asUS S Patent Appl. Publ. No.:20040192626) and various delivery approaches(see U.S. Ser. No. 11/586,102, published as US US-.S. Patent Appl. Publ.No. 20080020058)), which have provided compounds and compositionsavailable for clinical development. Nevertheless there remains a needfor additional RNA triggers that are available to expand the repertoireof available compounds and compositions for use in RNAi basedtherapeutics, and especially compounds and compositions that arecompatible with different delivery systems and/or routes ofadministration.

SUMMARY OF THE INVENTION

The invention provides a solution to the problem of having a sufficientrepertoire of available compounds and compositions for use in RNAi basedtherapeutics that are compatible with different delivery modalitiesand/or routes of administration by providing additional forms ofchemically modified short interfering nucleic acid (siNA) molecules.

The present invention provides compounds, compositions, and methodsuseful for modulating the expression of target genes and for treatingdiseases and conditions that respond to such modulation by RNAinterference (RNAi). Specially, the present invention provides certainchemically modified short interfering nucleic acid (siNA) molecules foruse as RNAi based therapeutic compounds and compositions.

In one embodiment, double-stranded short interfering nucleic acid (siNA)molecules are provided that modulate the expression of a target gene viaRNA interference, wherein the molecule has a sense strand and anantisense strand and comprises structure represented by formula (A);

-   -   wherein, the upper strand is the sense strand and the lower        strand is the antisense strand of the double-stranded nucleic        acid molecule; wherein the antisense strand comprises a sequence        having at least 15 nucleotides that are complementary to a        target RNA sequence encoded by the target gene and the sense        strand comprises a sequence that is complementarity to the        antisense strand;    -   each N is independently a nucleotide which is unmodified or        chemically modified or is optionally a non-nucleotide;    -   each B is independently a terminal cap that is present or        absent;    -   (N) represents overhanging nucleotides, each of which is        independently unmodified or chemically modified;    -   [N] represents nucleotides at the 5′-terminus of the antisense        strand;    -   X1 and X2 are independently integers from 0 to 4;    -   X3 is an integer from 15 to 30;    -   X4 is an integer from 12 to 27; and    -   X5 is an integer from 1-6, provided that the sum of X4 and X5 is        an integer from 15-30.

In a related embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) of formula (A); wherein

-   -   one or more pyrimidine nucleotides in N_(X4) positions are        independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl        nucleotides, 2′-deoxy nucleotides, ribonucleotides, or any        combination thereof;    -   one or more purine nucleotides in N_(X4) positions are        independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl        nucleotides, 2′-deoxy nucleotides, ribonucleotides, or any        combination thereof;    -   one or more pyrimidine nucleotides in N_(X3) positions are        independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl        nucleotides, 2′-deoxy nucleotides, ribonucleotides, or any        combination thereof;    -   one or more purine nucleotides in N_(X3) positions are        independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl        nucleotides, 2′-deoxy nucleotides, ribonucleotides; and    -   [N] position nucleotide(s) are ribonucleotides,        deoxyribonucleotides, 2′-O-alkyl nucleotides, 2′-halo        nucleotides, or any combination thereof irrespective of purine        or pyrimidine content.

In a related embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) of formula (A); wherein

-   -   5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides in N_(X4)        positions are 2′-O-alkyl nucleotides;    -   5, 6. 7 8, 9, 10 or more purine nucleotides in N_(X4) positions        are 2′-halo nucleotides;    -   5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides in N_(X3)        positions are 2′-O-alkyl nucleotides;    -   5, 6, 7, 8, 9, 10 or more purine nucleotides in N_(X3) positions        are 2′-halo nucleotides; and    -   [N] position nucleotide(s) are ribonucleotides,        deoxyribonucleotides, 2′-O-alkyl nucleotides, 2′-halo        nucleotides, or any combination thereof irrespective of purine        or pyrimidine content.

In a related embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) of formula (A); wherein

-   -   5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides in N_(X4)        positions are 2′-O-methyl nucleotides:    -   5, 6, 7, 8, 9, 10 or more purine nucleotides in N_(X4) positions        are 2′-deoxy-2′-fluoro nucleotides;    -   5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides in N_(X3)        positions are 2′-O-methyl nucleotides;    -   5, 6, 7, 8, 9, 10 or more purine nucleotides in N_(X3) positions        are 2′-deoxy-2′-fluoro nucleotides; and    -   [N] position nucleotide(s) are ribonucleotides,        deoxyribonucleotides, 2′-O-alkyl nucleotides, 2′-halo        nucleotides, or any combination thereof irrespective of purine        or pyrimidine content.

With respect to any siNA having Formula (A) described herein, in certainembodiments, X5=3, wherein the three [N] nucleotides of formula (A) arerepresented as 5′-[N1, N2, N3]-3′, wherein:

-   -   each N1, N2, and N3 is a ribonucleotide; or    -   each N1, N2, and N3 is a 2′-deoxy-2′-fluoro nucleotide; or    -   each N1, N2, and N3 is a 2′-deoxy nucleotide; or    -   each N1, N2, and N3 is a 2′-O-alkyl nucleotide; and    -   any of N1, N2, or N3 optionally comprises a phosphorothioate        internucleotide linkage.

With respect to any siNA having Formula (A) described herein, in certainembodiments, X5=3, wherein the three [N] nucleotides of formula (A) arerepresented as 5′-[N1, N2, N3]-3′, wherein:

-   -   N1 is a 2′-deoxy-2′-fluoro nucleotide, N2 is 2′-deoxy-2′-fluoro        nucleotide, and N3 is a 2′-deoxynucleotide; and    -   any of N1, N2, or N3 optionally comprises a phosphorothioate        internucleotide linkage.

With respect to any siNA having Formula (A) described herein, in certainembodiments, X5=3, wherein the three [N] nucleotides of formula (A) arerepresented as 5′-[N1, N2, N3]-3′, wherein:

-   -   N1 is a 2′-deoxy-2′-fluoro nucleotide, N2 is 2′-deoxy-2′-fluoro        nucleotide, and N3 is a 2′-deoxy-2′-fluoro nucleotide; and    -   any of N1, N2, or N3 optionally comprises a phosphorothioate        internucleotide linkage.

With respect to any siNA having Formula (A) described herein, in certainembodiments, X5=3, wherein the three [N] nucleotides of formula (A) arerepresented as 5′-[N1, N2, N3]-3′, wherein:

-   -   N1 is a 2′-deoxy nucleotide, N2 is 2′-deoxy-2′-fluoro        nucleotide, and N3 is a 2′-alkylnucleotide; and    -   any of N1, N2, or N3 optionally comprises a phosphorothioate        internucleotide linkage.

With respect to any siNA having Formula (A) described herein, in certainembodiments, X5=3, wherein the three [N] nucleotides of formula (A) arerepresented as 5′-[N1, N2, N3]-3′, wherein;

N1 is a 2′-deoxy nucleotide, N2 is 2′-deoxy-2′-fluoro nucleotide, and N3is a 2′-deoxy nucleotide; and

-   -   any of N1, N2, or N3 optionally comprises a phosphorothioate        internucleotide linkage.

With respect to any siNA having Formula (A) described herein, in certainembodiments, the siNA molecule is covalently attached to a polymer orligand via a linker. In certain embodiments, the siNA molecule iscovalently attached to the polymer or ligand via a linker moiety at the5′-end of the passenger (sense) strand of the siNA molecule. In otherembodiments, the siNA molecule is covalently attached to the polymer orligand via a linker moiety at the 3′-end of the passenger (sense) strandof the siNA molecule. In other embodiments, the siNA molecule iscovalently attached to the polymer or ligand via a linker moiety at the3′-end of the guide (antisense) strand of the siNA molecule. In any ofthe above embodiments, the linker can be attached to the terminal 3′and/or 5′ nucleotide position of the passenger or guide strand, or canalternately be attached to a terminal cap moiety such as an abasicmoiety or other cap as described herein or otherwise known in the art.Therefore, in totality, a siNA molecule of the invention having Formula(A) can comprise a terminal cap (B) that includes a covalent attachmentto a polymer or ligand via a linker molecule as described herein or asis otherwise known in the art. Non-limiting examples of such linkers areprovided in the examples herein.

In certain embodiments, one or more terminal cap moieties of a siNAmolecule of the invention (i.e. any B of any compound having Formula Aherein) can comprise a delivery modality. The delivery modality cancomprise a ligand or polymer that further includes one or more linkermolecules. Non-limiting examples of such linker molecules includephosphate ester based linkages, amino based linkers, disulfide basedlinkers, succinyl based linkers, alkyl or substituted alkyl basedlinkers, and/or amide based linkers as are generally known in the art.

In some embodiments, the siNA molecules of the invention arephosphorylated at the 5′ end of the antisense strand. The phosphategroup can be a phosphate, a diphosphate or a triphosphate.

The present invention further provides compositions comprising thedouble-stranded nucleic acid molecules described herein with optionallya pharmaceutically acceptable carrier or diluent.

In some embodiments, the invention features a composition comprising:

-   -   (a) a double-stranded short interfering nucleic acid (siNA) of        the invention; and    -   (b) a cationic lipid compond having any of compound numbers 1-46        or any combination thereof.

In some embodiments, the invention features a composition comprising:

-   -   (a) a double-stranded short interfering nucleic acid (siNA) of        the invention;    -   (b) a cationic lipid compond having any of compound numbers 1-46        or any combination thereof;    -   (c) cholesterol;    -   (d) DSPC; and    -   (e) PEG-DMG.

In some embodiments, the invention features a composition comprising:

-   -   (a) a double-stranded short interfering nucleic acid (siNA3 of        the invention;    -   (b) (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine;    -   (c) cholesterol;    -   (d) DSPC; and    -   (e) PEG-DMG.

In sonic embodiments, a composition of the invention comprises acationic lipid compond having any of compound numbers 1-46 (or anycombination thereof), cholesterol, and PECI-DMG (or alternatelyPEG-C-DMA) in the following molar ratios:

-   -   Cationic Lipid/Cholesterol/PEG-DMG 56.6/38/5.4;    -   Cationic Lipid/Cholesterol/PEG-DMO 60/38/2;    -   Cationic Lipid/Cholesterol/PEG-DMG 67.3/29/3.7;    -   Cationic Lipid/Cholesterol/PEG-DMG 49.3/47/3.7;    -   Cationic Lipid/Cholesterol/PEG-DMG 50.3/44.3/5.4;    -   Cationic Lipid/Cholesterol PEG-C-DMA/DSPC 40/48/2/10;    -   Cationic Lipid/Cholesterol/PEG-DMG/DSPC 40/48/2/10; and    -   Cationic Lipid/Cholesterol/PECI-DMG/DSPC 58/30/2/10.

In some embodiments, a composition of the invention comprises(13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, cholesterol,DSPC, and PEO-DMG, having a molar ratio of about 50:30:10:2respectively.

In some embodiments, a composition of the invention further comprises acryoprotectant. In some embodiments, the cryoprotectant is Sucrose,Trehalose, Raffinose, Stachyose, Verbascose, Mannitol, Glucose, Lactose,Maltose, Maltotriose-heptaose, Dextran, Hydroxyethyl Starch, Insulin,Sorbitol, Glycerol, Arginine, Histidine, Lysine, Proline,Dimethylsulfoxide or any combination thereof. In some embodiments, thecryoprotectant is Sucrose. In some embodiments, the cryoprotectant isTrehalose. In sonic embodiments, the cryoprotectant is a combination ofSucrose and Trehalose.

The present invention further provides a polymer comprising adouble-stranded short interfering nucleic acid (siNA) molecule of theinvention.

The present invention further provides a compound comprising adouble-stranded short interfering nucleic acid (siNA) molecule of theinvention covalently attached to a ligand. Non limiting examples ofligands include steroidal compounds (e.g., cholesterol), galactosaminesN-acetylgaiactosamine), vitamins (e.g., folate), proteins (e.g.,monoclonal antibodies), and peptides (e.g., TAT) as are generally knownin the art and further provided herein.

The present invention further provides a lipid nanoparticle (LNP)composition comprising the double-stranded short interfering nucleicacid (siNA) molecule of the invention. Non-limiting examples of LNPformulations are described herein and in PCT/US 11/52328, which isincorporated by reference herein in its entirely including the drawings.

The administration of the compositions of the invention can be carriedout by known methods, wherein the nucleic acid is introduced into adesired target cell in vitro or in vivo.

Commonly used techniques for introduction of the nucleic acid moleculesof the invention into cells, tissues, and organisms include the use ofvarious carrier systems, reagents and vectors. Non-limiting examples ofsuch carrier systems suitable for use in the present invention includesingle chemical entity conjugates, nucleic-acid-lipid particles, lipidnanoparticles (LNP), liposomes, lipoplexes, micelles, virosomes, viruslike particles (VLP), nucleic acid polymers, and mixtures thereof.

The compositions of the invention can be in the form of an aerosol,dispersion, solution (e.g., an injectable solution), a cream, ointment,tablet, powder, suspension or the like. These compositions may beadministered in any suitable way, e.g. orally, sublingually, buccally,parenterally, nasally, or topically, In some embodiments, thecompositions are aerosolized and delivered via inhalation.

The molecules and compositions of the present invention have utilityover a broad range of therapeutic applications. Accordingly anotheraspect of this invention relates to the use of the compounds andcompositions of the invention in treating a subject. The invention thusprovides a method for treating a subject, such as a human, sufferingfrom a condition which is associated with the expression of one or moregenes, wherein the method comprises administering to the subject aneffective amount of a double-stranded short interfering nucleic acid(siNA) molecule of the invention. Thus, the siNA molecules of theinvention treat the disease or condition. In some embodiments, thecondition is one as described herein or is otherwise generally known toone of skill in the art.

Additionally, the invention provides methods for stabilizing an siNAhave Formula A as defined above.

These and other aspects of the invention will be apparent upon referenceto the following detailed description and attached figures. Moreover, itis contemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein and that different embodiments may be combined.

Additionally, patents, patent applications, and other documents arecited throughout the specification to describe and more specifically setforth various aspects of this invention. Each of these references citedherein is hereby incorporated by reference in its entirety, includingthe drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting proposed mechanistic representation oftarget RNA degradation involved in RNAi. Double-stranded RNA (dsRNA),which is generated by RNA-dependent RNA polymerase (RdRP) from foreignsingle-stranded RNA, for example viral, transposon, or other exogenousRNA, activates the DICER enzyme that in turn generates siNA duplexes.Alternately, synthetic or expressed siNA can be introduced directly intoa cell by appropriate means. An active siNA complex forms thatrecognizes a target. RNA, resulting in degradation of the target RNA bythe RISC endonuclease complex or in the synthesis of additional RNA byRNA-dependent RNA polymerase (RdRP), which can activate DICER and resultin additional siNA molecules, thereby amplifying the RNAi response.

FIGS. 2A and 2B show non-limiting examples of chemically modified siNAconstructs of the present invention using a generalized structure of arepresentative siNA duplex, The specific modifications shown in thefigure can be utilized alone or in combination with other modificationsof the figure, in addition to other modifications and features describedherein with reference to any siNA molecule of the invention. In FIG. 2A,N stands for any nucleotide or optionally a non-nucleotide as describedhere. The upper strand, having B—N_(X3)—(N)_(X2)—B-3′ is the sense (orpassenger) strand of the siNA, whereas the lower strand, havingB(N)_(X1)—N_(X4)—[N]_(X5)-5′ is the antisense (or guide) strand of thesiNA, Nucleotides (or optional non-nucleotides) of internal portions ofthe sense strand are designated N_(X3) and nucleotides (or optionalnon-nucleotides) of internal portions of the antisense strand aredesignated N_(X4). Nucleotides (or optional non-nucleotides) of theinternal portions are generally base paired between the two strands, butcan optionally lack base pairing (e.g. have mismatches or gaps) in someembodiments. Nucleotides (or optional non-nucleotides) of overhangregions are designated by parenthesis (N), Nucleotides of the5′-terminal portion of the antisense strand are designated [N]. Terminalcaps are optionally present at the 5′ and/or 3′ end of the sense strandand further optionally present at the 3′-end of the antisense strand.Generally, each strand can independently range from about 15 to about 30nucleotides in length, but can vary depending on the presence of anyoverhang nucleotides. In certain embodiments, X1 and X2 areindependently integers from 0 to 4; X3 is an integer from 15 to 30; X4is an integer from 9 to 30; X5 is an integer from 0 to 6, provided thatthe sum of X4 and X5 is 15-30. Various modifications are shown for thenucleotides of the sense and antisense strands of the siNA constructs.The (N) overhang nucleotide positions can be chemically modified asdescribed herein (e.g., 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-deoxy, LNA,universal bases etc.) and can be either derived from a correspondingtarget nucleic acid sequence or not. The constructs shown in the figurecan also comprise phosphorothioate linkages as described herein, Forexample, phosphorothioate linkages can exist between any N, (N), and/or[N] positions. Such phosphorothioate incorporation can be utilizedbetween purine “R” and pyrimidine “Y” positions, or for stabilization ofpyrimidine linkages in general. Furthermore, although not depicted onthe figure, the constructs shown in the figure can optionally include aribonucleotide at the 9^(th) position from the 5′-end of the sensestrand or the 11^(th) position based on the 5′-end of the guide strandby counting 11 nucleotide positions in from the 5′-terminus of the guidestrand. Similarly, the antisense strand can include a ribonucleotide ora 2′-deoxy-2′-fluoro nucleotide at the 14^(th) position from the 5′-end,or alternately can be selected or designed so that a 2′-O-alkylnucleotide (e.g., a 2′-O-methyl purine) is not present at this position.Furthermore, although not shown in the figure, the 5′-terminal positionof the antisense strand can comprise a terminal phosphate group asdescribed herein. The antisense strand generally comprises sequencecomplementary to any target nucleic acid sequence of the invention. InFIG. 2B, N stands for any nucleotide or optionally a non-nucleotide asdescribed herein. The upper strand, having B—N_(X3)—(N)_(X2)—B-3′ is thesense (or passenger) strand of the siNA, whereas the lower strand,having B(N)_(X1)—N_(X4)—[N3]-[N2]-[N1]-5 is the antisense (or guide)strand of the siNA. Nucleotides (or optional non-nucleotides) ofinternal portions of the sense strand are designated N_(X3) andnucleotides (or optional non-nucleotides) of internal portions of theantisense strand are designated N_(S4). Nucleotides (or optionalnon-nucleotides) of the internal portions are generally base pairedbetween the two strands, but can optionally lack base pairing (e.g. havemismatches or gaps) in some embodiments. Nucleotides (or optionalnon-nucleotides) of overhang regions are designated by parenthesis (N).Nucleotides of the 5′-terminal portion of the antisense strand aredesignated [N]. Terminal caps are optionally present at the 5′ and/or 3′end of the sense strand and further optionally present at the 3′-end ofthe antisense strand. Generally, each strand can independently rangefrom about 15 to about 30 nucleotides in length, but can vary dependingon the presence of any overhang nucleotides. In certain embodiments, X1and X2 are independently integers from 0 to 4; X3 is an integer from 15to 30; and X4 is an integer from 12 to 27. Various modifications areshown for the nucleotides of the sense and antisense strands of the siNAconstructs. The [N3], [N2], and [N1] nucleotides are chemically modifiedwith either 2′-deoxy, 2′-deoxy-2′-fluoro or 2′-methoxy modifications.The (N) overhang nucleotide positions can be chemically modified asdescribed herein (e.g., 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-deoxy, LNA,universal bases etc.) and can be either derived from a correspondingtarget nucleic acid sequence or not. The constructs shown in the figurecan also comprise phosphorothioate linkages as described herein. Forexample, phosphorothioate linkages can exist between any N, and/or (N)positions. Such phosphorothioate incorporation can be utilized betweenpurine “R” and pyrimidine “Y” positions, or for stabilization ofpyrimidine linkages in general. Furthermore, although not depicted onthe figure, the constructs shown in the figure can optionally include aribonucleotide at the 9^(th) position from the 5′-end of the sensestrand or the 11^(th) position based on the 5′-end of the guide strandby counting 11 nucleotide positions in from the, 5′-terminus of theguide, strand. Similarly, the antisense strand can include aribonucleotide; or a 2′-deoxy-2′-fluoro nucleotide at the 14^(th)position from the 5′ end, or alternately can be selected or designed sothat a 2′-O-alkyl nucleotide (e.g., a 2′-O-methyl purine) is not presentat this position. Furthermore, although not shown in the figure, the5′-terminal position of the antisense strand can comprise a terminalphosphate group as described herein.

FIG. 3 shows non-limiting examples of certain combinations ofmodifications applied to the representative siNA duplex described inFIG. 2A. The table shown below the representative structure providesspecific combinations of (N)_(X1), (N)_(X2), N_(X3), N_(X4), and/or[N]x5 nucleotide (and optional non-nucleotide) positions. For example,combinations of 5 or more (e.g., 5, 6, 7, 8, 9, or 10 or more) N_(X3)and 5 or more (e.g., 5, 6, 7, 8, 9, or 10 or more) N_(X4) pyrimidine “Y”and purine “R” nucleotides are specified, each of which canindependently have specific (N)_(X1), and/or (N)_(X2), substitutions asshown in the figure, in addition to optional phosphorothioatesubstitutions. The 5′-terminal antisense strand [N] nucleotides aregenerally ribonucleotides, but can also be modified or unmodifieddepending on if they are purine “R” or pyrimidine “Y” nucleotides

FIG. 4 shows additional non-limiting examples of certain combinations ofmodifications applied to the representative siNA duplex described inFIG. 2B and having specific combinations of 5′-guide strandmodifications. The table shown below the representative structureprovides specific combinations of (N)_(X1), (N)_(X2), N_(X3), N_(X4),and [N3]-[N2]-[N1] nucleotide (and optional non-nucleotide) positions.For example, combinations of 5 or more (e.g., 5, 6, 7, 8, 9, or 10 ormore) N_(X3) and 5 or more (e.g., 5, 6, 7, 8, 9, or 10 or more) N_(X4)pyrimidine “Y” and purine “R” nucleotides are specified, each of whichcan independently^(,) have specific (N)_(X1), and/or (N)_(X2),substitutions as shown in the figure, in addition to optionalphosphorothioate substitutions. The 5′-terminal antisense strand[N3]-[N2]-[N1] nucleotides are modified with either 2′-deoxy,2′-deoxy-2′-fluoro or 2′-methoxy modifications as depicted.

FIG. 5A-C shows non-limiting examples of different siNA constructs ofthe invention. The criteria of the representative structures shown inFIGS. 2A, 2B, 3 and 4 can be applied to any of the structures shown inFIG. 5A-C.

The examples shown in FIG. 5A (constructs 1, 2, and 3) have 19representative base pairs; however, different embodiments of theinvention include any number of base pairs described herein. Bracketedregions represent nucleotide overhangs, for example, comprising about 1,2, 3, or 4 nucleotides in length, preferably about 2 nucleotides.Constructs 1 and 2 can be used independently for RNAi activity.Construct:2 can comprise a polynucleotide or non-nucleotide linker,which can optionally be designed as a biodegradable linker. In oneembodiment, the loop structure shown in construct 2 can comprise abiodegradable linker that results in the formation of construct 1 invivo and/or in vitro. In another example, construct 3 can be used togenerate construct 2 under the same principle wherein a linker is usedto generate the active siNA construct 2 in vivo and/or in vitro, whichcan optionally utilize another biodegradable linker to generate theactive siNA construct 1 in vivo and/or in vitro. As such, the stabilityand/or activity of the siNA constructs can be modulated based on thedesign of the siNA construct for use in vivo or in vitro and/or invitro.

The examples shown in FIG. 5B represent different variations ofdouble-stranded nucleic acid molecule of the invention, such asmicroRNA, that can include overhangs, bulges, loops, and stem-loopsresulting from partial complementarity. Such motifs having bulges,loops, and stem-loops are generally characteristics of miRNA. Thebulges, loops, and stem-loops can result from any degree of partialcomplementarity, such as mismatches or bulges of about 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more nucleotides in one or both strands of thedouble-stranded nucleic acid molecule of the invention.

The example shown in FIG. 5C represents a model double-stranded nucleicacid molecule of the invention comprising a 19 base pair duplex of two21 nucleotide sequences having dinucleotide 3′-overhangs. The top strand(1) represents the sense strand (passenger strand), the middle strand(2) represents the antisense (guide strand), and the lower strand (3)represents a target polynucleotide sequence, The dinucleotide overhangs(NN) can comprise a sequence derived from the target polynucleotide. Forexample, the 3′-(NN) sequence in the guide strand can be complementaryto the 5′-[NN] sequence of the target polynucleotide. In addition, the5′-(NN) sequence of the passenger strand can comprise the same sequenceas the 5′-[NN] sequence of the target polynucleotide sequence. In otherembodiments, the overhangs (NN) are not derived from the targetpolynucleotide sequence, for example where the 3′-(NN) sequence in theguide strand are not complementary to the 5′-[NN] sequence of the targetpolynucleotide and the 5′-(NN) sequence of the passenger strand cancomprise different sequence from the 5′-[NN] sequence of the targetpolynucleotide sequence. In additional embodiments, any (NN) nucleotidesare chemically modified, e.g., as 2′-O-methyl, 2′-deoxy-2′-fluoro,and/or other modifications herein, Furthermore, the passenger strand cancomprise a ribonucleotide position N of the passenger strand. For therepresentative 19 base pair 21 mer duplex shown, position N can be 9nucleotides in from the 5′ end of the passenger strand. However, induplexes of differing length, the position N is determined based on the5′-end of the guide strand by counting 11 nucleotide positions in fromthe 5′-terminus of the guide strand and picking the corresponding basepaired nucleotide in the passenger strand. Cleavage by Ago2 takes placebetween positions 10 and 11 as indicated by the arrow. In additionalembodiments, there are two ribonucleotides, NN, at positions 10 and 11based on the 5′-end of the guide strand by counting 10 and 11 nucleotidepositions in from the 5′-terminus of the guide strand and picking thecorresponding base paired nucleotides in the passenger strand. Theantisense strand nucleotide N can also be a ribonucleotide or modifiednucleotide and is located at position 14 from the 5′-end terminus of theguide strand. The modification can be, for example, a 2′-deoxy-2′-fluoromodification, but is preferably not a 2′-O-alkyl modification. PositionN3, N2, and N1 of the antisense strand comprise modified nucleotides.

FIG. 6 shows non-limiting examples of different stabilizationchemistries (1-10) that can be used, for example, to stabilize the 5′and/or 3′-ends of siNA sequences of the invention, including (1)[3-3′]-inverted deoxyribose; (2) deoxyribonucleotide; (3)[5′-3]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5)[5′-3′]-3′-O-methyl ribonucleodde; (6) 3′-glyceryl; (7) [3′-5′]-3′-deoxyribonucleotide ; (8) [3′- 3′]-deoxyribonucleotide; (9)[5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide (whenX=O). In addition to modified and unmodified backbone chemistriesindicated in the figure, these chemistries can be combined withdifferent sugar and base nucleotide modifications as described herein.

FIG. 7 shows non-limiting examples of phosphorylated siNA molecules ofthe invention, including linear and duplex constructs and asymmetricderivatives thereof.

FIG. 8 shows non-limiting examples of chemically modified terminalphosphate groups of the invention.

FIG. 9 shows a non-limiting example of a cholesterol linkedphosphoramidite that can be used to synthesize cholesterol conjugatedsiNA molecules of the invention. An example is shown with thecholesterol moiety linked to the 5′-end of the sense strand of an siNAmolecule.

FIG. 10 depicts an embodiment of 5′ and 3′ inverted abasic caps linkedto a nucleic acid strand. These inverted abasic caps can be derivatizedwith linker molecules to serve as points of attachment of the siNAmolecules of the invention to polymer or ligand based delivery systems.For example, the terminal hydroxyl group present on an inverted abasicmoiety, such as at the 5′-end, 3′-end, or both 5′ and 5′-ends of one orboth strands of the siNA molecule of the invention, can be conjugatedvia a linker molecule (as described herein or as otherwise known in theart) to a ligand delivery modality (e.g., a steroid such as cholesterol,an antibody, a vitamin such as folate, a galactosamine moiety such asN-acetylgalactosamine (NAG), or a peptide such as TAT) or to a polymericdelivery modality as described herein or as otherwise known in the art.Therefore, in certain embodiments, one or more terminal cap moieties ofa siNA molecule of the invention (i.e. any B of a compound havingFormula A herein) can comprise a delivery modality. The deliverymodality can comprise a ligand or polymer that further includes one ormore linker molecules (e.g., a phosphate ester based linkage, an aminobased linker, a disulfide based linker, a succinyl based linker, analkyl or substituted alkyl based linker, or an amide based linker).

FIG. 11 depicts in vitro serum stability and mRNA knockdown at 10 nM orApoB (9514) siRNAs with varied chemical modifications but commonunderlying sequence. The stab07 passenger strand is composed of 2′F(2′-deoxy-2′-fluoro) pyrimidines and 2′H (2′-deoxy) purines and containsinverted abasic caps on 5′ and 3′ ends. The stab07H passenger simplyadds is phosphorothioate linkage between positions 20 and 21. The stab35guide strand is unmodified at positions 1-3 with the remainder of strandcomposed of 2′OMe (2′-O-methyl) purines and 2′F (2′-deoxy-2′-fluoro)pyrimidines. The stab35N modification motif adds a phosphorothioatelinkage between positions 20-21 while the stab35U2 motif additionallyhas phosphorothioate linkages at positions 1-3. The RNASci10modification motif differs from stab07/35 motifs in that pyrimidines are2′OMe modified while purines are 2′F. Note for all guide strands thatposition 14 is a 2′F regardless of pyrimidine or purine identity. SeeFIG. 12 for details. mRNA knockdown is measured in mouse Hepa1-6 cellsusing RNAiMax transfection reagent and an siRNA concentration of 10 nM.In vitro serum stability is measured by mass spectrometry and expressedas a percentage of the fully intact parental strand.

FIG. 12 depicts an evaluation of the tolerance of 2′-ribose sugarmodifications at position 14 of the siRNA guide strand. 2′F, 2′OMe, and2′H ribose modifications were tested in seven different siRNA sequences.Knockdown for individual siRNAs are shown and the horizontal linerepresents the median knockdown for the seven siRNAs tested, mRNAknockdown was measured as a log 2 fold-change relative to the parentalunmodified siRNA sequence with negative values indicating a deleteriouseffect activity. Position 14 is largely intolerant of 2′OMe substitutionwhile 2′F is best tolerated.

FIGS. 13A and 13B depict in vivo data for APoB siRNA with the Sci10modification motif. mRNA knockdown is shown as log 2 fold change inliver mRNA expression with negative values indicating a greater amountof siRNA knockdown. ApoB liver mRNA expression was measured byquantitative RT-PCR. siRNAs were delivered to mice using the polymerconjugate delivery vehicle. Knockdown is compared at day 2 and day 7timepoints (FIG. 13A) and days 2, 7, 14, 21 (FIG. 13B). Relative to the07/35 modification motifs, the Sci10 modification motif has greaterinitial knockdown and longer duration of activity.

FIGS. 14A and 14B depict data demonstrating that the Sci10 modificationmotif is compatible with polymer conjugate (FIG. 14A) and lipidnanoparticle (FIG. 14B) delivery vehicles in vivo. ApoB mRNA expressionwas measured from mouse livers as discussed with respect to FIGS. 13Aand 13B. The composition of the lipid nanoparticle shields the siRNAcargo from serum nucleases and therefore the 07H/35N modification motifis equivalently active to the Sci10 motif with this particular LNPdelivery platform. Note that the LNP delivered siRNAs (FIG. 14B) differslightly from the PC delivered siRNAs (FIG. 14A). The siRNAs in (FIG.14B) do not contain the amino linker (6amiL) which is used to conjugatethe siRNA to the PC delivery vehicle.

FIG. 15 depicts a comparison of the in vitro mRNA knockdown and serumstability for ApoB Sci10 and modified variants. These modified variantsreplace the three phosphorothioate linkages at the 5′ of the guidestrand with specific combinations of 2′ ribose sugar modifications topositions 1-3 of the guide. The Sci10 modification motif and details ofknockdown and stability measurements are described in FIG. 11. Thevariants to position 1-3 of the guide strand are: “Sci10iff”representing 213′F at positions 1-3; “Sci10ffd” representing 2′F atpositions 1-2 and 2′H at position 3; “Sci10dfd” representing 2′H atposition 1, 2′F at position 2, and 2′H at position 3; “Sci10dfm”representing 2′H at position 1, 2′F at position 2., and 2′OMe atposition 3. The Sci10 variants have vitro mRNA knockdown and serumstability comparable to the Sci10 modification motif containingphosphorothioates at the 5′ of the guide strand.

FIG. 16 depicts that 5′-guide strand modified variants of the Sci10modification motif possess equivalent in vivo duration of mRNA knockdownrelative to the Sci10 modification motif with phosphorothioatemodifications at the 3 terminal 5′-guide strand positions. siRNAs weredelivered with polymer conjugate and ApoB mRNA expression was measuredfrom mouse livers as discussed in FIGS. 13A and 13B. FIG. 15 details thein vitro knockdown and stability for these siRNAs.

FIG. 17 depicts that the Sci10 modification motif can be applied toother sequences of interest, such as SSB (291), while retaining in vitromRNA knockdown activity while significantly improving serum nucleasestability. The 07H/35N siRNA has moderate passenger strand stability andlittle observable guide strand stability. The Sci10 modification motifcontaining 5′ guide strand phosphorothioate linkages improves passengerstrand stability but has no affect on the guide strand. The “YA” atpositions 2-3 of the siRNA guide strand indicate the. presence of apyrimidine-adenosine motif which is known to be highly susceptible tonuclease cleavage. Replacement of the 5′ phosphorothioates with thevariants described in FIG. 15 results in significantly improved guidestrand stability while retaining mRNA activity. The 07/35 and. Sci10modification motifs and details of knockdown and stability measurementsare described in FIG. 11.

FIG. 18 depicts data in which SSB (291) siRNAs with the Sci10dfm andSci10ffd motifs were compared against the 07H/35N motif in vivo forduration of snRNA knockdown. The Sci10 modification motifs exceed07H/35N in initial knockdown and duration of the. knockdown effect.siRNAs were delivered with polymer conjugate and SSB mRNA expression wasmeasured from mouse livers using methods discussed in FIGS. 13A and 13B.

FIGS. 19A and 19B depict that 2′F content in the Sci10 modificationmotif confers improved in vivo duration of mRNA knockdown. (FIG. 19A)ApoB (9514) Sci10 modification motif is compared to Sci11 (2′F purinesin passenger strand are changed to 2′OH) and Sci07f (2′F purines in bothpassenger and guide strand are changed to 2′OH). Overall in vitroknockdown and stability levels are similar among these siRNAs, thoughSci11 has slightly reduced stability. Details of knockdown and stabilitymeasurements are discussed in FIG. 11. (FIG. 19B) Duration of in vivoknockdown is compared for Sci10, Sci11 and Sci07f. Sci11 hasintermediate activity and less duration than Sci10 and Sci07f has nosignificant mRNA knockdown. This graded response appears to correlatewith the amount of 2′F content present in the siRNA. siRNAs weredelivered with polymer conjugate and ApoB mRNA expression was measuredfrom mouse livers as discussed in FIGS. 13A and 13B. (FIG. 19C)Comparison of the in vivo liver metabolism of Sci10, Sci11, and Sci07fsiRNAs at 48 hours. The FDA defines a major metabolite as “those formedat greater than 10 percent of parent drug systemic exposure at steadystate”. Therefore major metabolites are defined as >10% of parent strandat 48 hours and minor metabolites are defined as <10% of parent strandat 48 hours. Sci10 shows only minor metabolism sites while Sci11 andSci07f have sites of major cleavage suggesting 2′F content improvesintracellular stability.

FIGS. 20A and 20B depict that 2′F content is also important for the invivo duration of other siRNAs containing the Sci10 modification motif.(FIG. 20A) Comparison of variants of Sci10 (see FIG. 17) which have 2′Fpurine content and siRNAs which the 2′F purines are instead 2′OH (Sci.07variants). Details of knockdown and stability measurements are discussedin FIG. 11. (FIG. 20B) Sci10 modifications have significant duration ofmRNA knockdown over 21 days while the Sci07 variants have significantlyreduced duration. As seen for ApoB (FIG. 19B) this suggests that 2′Fcontent can be important for duration of siRNA knockdown in vivo. siRNAswere delivered with polymer conjugate and SSB mRNA expression wasmeasured from mouse livers using methods discussed in FIGS. 13A and 13B.

DETAILED DESCRIPTION OF THE INVENTION A. Terms and Definitions

The following terminology and definitions apply as used in the presentapplication.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a cell”includes a combination of two or more cells, and the like.

Any concentration range, percentage range, ratio range or integer rangeis to be understood to include the value of any integer within therecited range, and when appropriate, fractions thereof (such as on tenthand one hundredth of an integer), unless otherwise indicated.

“About” or “approximately,” as used herein , in reference to a numberare generally taken to include numbers that fall within a range of 5% ineither direction (greater than or less than) of the number unlessotherwise stated or otherwise evident from the context (except wheresuch number would exceed 100% of a possible value). Where ranges arestated, the endpoints are included within the range unless otherwisestated or otherwise evident from the context.

The term “abasic” as used herein refers to its meaning as is generallyaccepted in the. art. The term generally refers to sugar moietieslacking a nucleobase or having a hydrogen atom (H) or othernon-nucleobase chemical groups in place of a nucleobase at the rposition of the sugar moiety, see for example Adaraic et al., U.S. Pat.No. 5,998,203. in one embodiment, an abasic moiety of the invention is aribose, deoxyribose, or dideoxyribose sugar.

The term “acyclic nucleotide” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to anynucleotide haying an acyclic ribose sugar, for example where any of theribose carbon/carbon or carbon/oxygen bonds are independently or incombination absent from the nucleotide.

The term “alkyl” as used herein refers to its meaning as is generallyaccepted in the art. The term generally refers to a saturated orunsaturated hydrocarbons, including straight-chain, branched-chain,alkenyl, alkynyl groups and cyclic groups, but excludes aromatic groups.Notwithstanding the foregoing, alkyl also refers to non-aromaticheterocyclic groups. Preferably, the alkyl group has 1 to 12 carbons.More preferably, it is a lower alkyl of from 1 to 7 carbons, morepreferably 1 to 4 carbons. The alkyl group can be substituted orunsubstituted. When substituted, the substituted group(s) is preferably,hydroxyl, halogen, cyano, C1—C4 alkoxy, ═O, ═S, NO₂ , SH, NH₂, or NR₁R₂,where R₁ and R2 independently are H or C1—C4 alkyl.

The phrase “agents that interfere with cell cycle checkpoints” refers tocompounds that inhibit protein kinases that transduce cell cyclecheckpoint signals, thereby sensitizing the cancer cell to DNA damagingagents.

The phrase “agents that interfere with receptor tyrosine kinases (RTKs)”refers to compounds that inhibit RTKs and therefore inhibit mechanismsinvolved in oncogenesis and tumor progression.

The phrase “androgen receptor modulators” refers to compounds thatinterfere or inhibit the binding of androgens to the receptor,regardless of mechanism.

The phrase “angiogenesis inhibitors” refers to compounds that inhibitthe formation of new blood vessels, regardless of mechanism.

The term “aryl” as used herein refers to its meaning as is generallyaccepted in the art. The term generally refers to an aromatic group thathas at least one ring having a conjugated pi electron system andincludes carbocyclic aryl, heterocyclic aryl and biaryl groups, all ofwhich can be optionally substituted. The preferred substituent(s) ofaryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, C1—C4alkoxy, C1—C4 alkyl, C2—C4 alkenyl, C2—C4 alkynyl, NH₂, and NR₁ R₂groups, where R₁ and R₂ independently are H err C1—C4 alkyl.

The term “alkylaryl” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to an alkylgroup (as described above) covalently joined to an aryl group (asdescribed above). Carbocyclic aryl groups are groups wherein the ringatoms on the aromatic ring are all carbon atoms. The carbon atoms areoptionally substituted. Heterocyclic aryl groups are groups having from1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainderof the ring atoms are carbon atoms. Suitable heteroatoms include oxygen,sulfur, and nitrogen, and examples of heterocyclic aryl groups havingsuch heteroatoms include furanyl, thienyl, pyridyl, pyrrolyl, N-loweralkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, alloptionally substituted. Preferably, the alkyl group is a C1—C4 alkylgroup.

The term “amide” as used herein refers to its meaning as is generallyaccepted in the art. The term generally refers to an —C(O)—NH—R, where Ris either alkyl, aryl, alkylaryl or hydrogen.

The phrase “antisense region” as used herein refers to its meaning as isgenerally accepted in the art. With reference to exemplary nucleic acidmolecules of the invention, the term refers to a nucleotide sequence ofan siNA molecule having complementarity to a target nucleic acidsequence. In addition, the antisense region of an siNA molecule canoptionally comprise a nucleic acid sequence having complementarity to asense region of the siNA molecule. In one embodiment, the antisenseregion of the siNA molecule is referred to as the antisense strand orguide strand.

The phrase “asymmetric hairpin” refers to a linear siNA moleculecomprising an antisense region, a loop portion that can comprisenucleotides or non-nucleotides, and a sense. region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complementary nucleotides to base pair with theantisense region and form a duplex with loop. For example, an asymmetrichairpin siNA molecule of the invention can comprise an antisense regionhaving length sufficient to mediate RNAi in a cell or in vitro system(e.g. about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 nucleotides) and a loop region comprisingabout 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or 12)nucleotides, and a sense region having about 3 to about 25 (e.g., about3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25) nucleotides that are complementary to the antisenseregion. The asymmetric hairpin siNA molecule can also comprise a5′-terminal phosphate group that can be chemically modified. The loopportion of the asymmetric hairpin siNA molecule can comprisenucleotides, non-nucleotides, linker molecules, or conjugate moleculesas described herein.

The term “biodegradable” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to degradationin a biological system, for example, enzymatic degradation or chemicaldegradation.

The term “biodegradable linker” as used herein refers to its meaning asis generally accepted in the art. With reference to exemplary nucleicacid molecules of the invention, the term refers to a linker moleculethat is designed to connect one molecule to another molecule, and whichis susceptible to degradation in a biological system. The linker can bea nucleic acid or non-nucleic: acid based linker. For example, abiodegradable linker can be used to attach a ligand or biologicallyactive molecule to an siNA molecule of the invention. Alternately, abiodegradable linker can be used to connect the sense and antisensestrands of an siNA molecule of the invention. The biodegradable linkeris designed such that its stability can be modulated for a particularpurpose, such as delivery to a particular tissue or cell type. Thestability of a nucleic acid-based biodegradable linker molecule can bemodulated by using various chemistries, for example combinations ofribonucleotides, deoxyribonucleotides, and chemically modifiednucleotides, such as 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino,2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modifiednucleotides. The biodegradable nucleic acid linker molecule can be adimer, trimer, tetramer or longer nucleic acid molecule, for example, anoligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 nucleotides in length, or can comprise a singlenucleotide with a phosphorus-based linkage, for example, aphosphoramidate or phosphodiester linkage. The biodegradable nucleicacid linker molecule can also comprise nucleic acid backbone, nucleicacid sugar, or nucleic acid base modifications.

The phrase “biologically active molecule” as used herein refers to itsmeaning as is generally accepted in the art. With reference to exemplarynucleic acid molecules of the invention, the term refers to compounds ormolecules that are capable of eliciting or modifying a biologicalresponse in a system and/or are capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules. Examples of biologically active molecules, include siNAmolecules alone or in combination with other molecules including, butnot limited to therapeutically active molecules such as antibodies,cholesterol, hormones, antivirals, peptides, proteins,chemotherapeutics, small molecules, vitamins, co-factors, nucleosides,nucleotides, oligonucleotides, enzymatic nucleic acids, antisensenucleic acids, triplex forming oligonucleotides, polyamines, polyamides,polyethylene glycol, other polyethers, 2-5A chimeras, siNA, dsRNA,allozymes, aptamers, decoys and analogs thereof.

The phrase “biological system” as used herein refers to its meaning asis generally accepted in the art. The term generally refers to material,in a purified or unpurified form, from biological sources including, butnot limited to, human or animal, wherein the system comprises thecomponents required for RNAi activity. Thus, the phrase includes, forexample, a cell, tissue, subject, or organism, or extract thereof. Theterm also includes reconstituted material from a biological source.

The phrase “blunt end” as used herein refers to its meaning as isgenerally accepted in the art. With reference to exemplary nucleic acidmolecules of the invention, the term refers to termini of adouble-stranded siNA molecule having no overhanging nucleotides. Forexample, the two strands of a double-stranded siNA molecule having bluntends align with each other with matched base-pairs without overhangingnucleotides at the termini. A siNA duplex molecule of the invention cancomprise blunt ends at one or both termini of the duplex, such astermini located at the 5′-end of the antisense strand, the 5′-end of thesense strand, or both termini of the duplex.

The terra “cap” also referred to herein as “terminal cap,” as usedherein refers to its meaning as is generally accepted in the art. Withreference to exemplary nucleic acid molecules of the invention, the termrefers to a moiety, which can be a chemically modified nucleotide ornon-nucleotide that can be incorporated at one or more termini of one ormore nucleic acid molecules of the invention. These terminalmodifications protect the nucleic acid molecule from exonucleasedegradation, and can help in delivery and/or localization within a cell.The cap can be present at the 5′-terminus (5′-cap) or at the 3′-terminal(3′-cap) or can be present on both termini of any nucleic acid moleculeof the invention. A cap can be present at the 5′-end, 3-end and/or 5′and 3′-ends of the sense strand of a nucleic acid molecule of theinvention. Additionally, a cap can optionally be present at the 3′-endof the antisense strand of a nucleic acid molecule of the invention. Innon-limiting examples, the 5′-cap includes, but is not limited to apolymer; a ligand; locked nucleic acid (1.NA); glyceryl; an abasicribose residue (moiety); inverted deoxy abasic residue (moiety); aninverted nucleotide; 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclicnucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;three-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide;3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexyiphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methyiphosphonate moiety, Non-limiting examplesof the 3′-cap include, but are not limited to, a polymer; a ligand;locked nucleic acid (LNA); glyceryl; an abasic ribose residue (moiety);inverted deoxy abasic residue (moiety); an inverted nucleotide; 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thionucleotide; carbocyclic nucleotide; 5′-amino-alkyl phosphate;1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexylphosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate;1,5-anhydrohexitol nucleotide; nucleotide; alpha-nucleotide; modifiedbase nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide;acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide;3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety;5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate;1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging5′-phosphoramidote: phosphorothioate and/or phosphorodithioate; bridgingor non bridging methylphosphonate; and 5′-mercapto moieties (for moredetails see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporatedby reference herein). In certain embodiments, a siNA molecule of theinvention having Formula (A) can comprise one or more terminal capmolecules as described above (designated as B) that comprises orincludes a covalent attachment to a polymer or ligand via a linkermolecule as described herein or as is otherwise known in the art.Non-limiting examples of such linkers are provided in the examplesherein. FIGS. 6 and 10 show some non-limiting examples of various caps.

The term “cell” as used herein refers to its meaning as is generallyaccepted in the art. With reference to exemplary nucleic acid moleculesof the invention, the term is used in its usual biological sense, anddoes not refer to an entire multicellular organism, e.g., specificallydoes not refer to a human being. The cell can be present in an organism,e.g., birds, plants and mammals, such as humans, cows, sheep, apes,monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g.,bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cellcan be of somatic or germ line origin, totipotent or pluripotent,dividing or non-dividing. The cell can also be derived from or cancomprise a gamete or embryo, a stem cell, or a fully differentiatedcell.

The phrase “chemical modification” as used herein refers to its meaningas is generally accepted in the art. With reference to exemplary nucleicacid molecules of the invention, the term refers to any modification ofthe chemical structure of the nucleotides that differs from nucleotidesof native siRNA or RNA in general. The term “chemical modification”encompasses the addition, substitution, or modification of native siRNAor RNA at the sugar, base, or internucleotide linkage, as describedherein or as is otherwise known in the art. in certain embodiments, theterm “chemical modification” can refer to certain forms of RNA that arenaturally occurring in certain biological systems, for example2′-O-methyl modifications or inosine modifications.

The term “complementarity” or “complementary” as used herein refers toits meaning as is generally accepted in the art. With reference toexemplary nucleic acid molecules of the invention, the terms generallyrefer to the formation or existence of hydrogen bond(s) between onenucleic acid sequence and another nucleic acid sequence by eithertraditional Watson-Crick or other non-traditional types of bonding asdescribed herein. In reference to the nucleic molecules of the presentinvention, the binding free energy for a nucleic acid molecule with itscomplementary sequence is sufficient to allow the relevant function ofthe nucleic acid to proceed, e.g., RNAi activity. Determination ofbinding free energies for nucleic acid molecules is well known in theart (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LIIpp.123-133; Prier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377;Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). Perfectcomplementary means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence. Partial complementarily can includevarious mismatches or non-based paired nucleotides (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more mismatches, non-nucleotide linkers, or non-basedpaired nucleotides) within the nucleic acid molecule, which can resultin bulges, loops, or overhangs that result between the sense strand orsense region and the antisense strand or antisense region of the nucleicacid molecule or between the antisense strand or antisense region of thenucleic acid molecule and a corresponding target nucleic acid molecule.Such partial complementarity can be represented by a % complementaritythat is determined by the number of non-base paired nucleotides, i.e.,about 50%, 60%, 70%, 80%, 90% etc. depending on the total number ofnucleotides involved. Such partial complementarity is permitted to theextent that the nucleic acid molecule (e.g., siNA) maintains itsfunction, for example the ability to mediate sequence specific RNAi.

The terms “composition” or “formulation” as used herein refer to theirgenerally accepted meaning in the art. These terms generally refer to acomposition or formulation, such as in a pharmaceutically acceptablecarrier or diluent, in a form suitable for administration, e.g.,systemic or local administration, into a cell or subject, including, forexample, a human. Suitable forms, in part, depend upon the use or theroute of entry, for example oral, transdermal, inhalation, or byinjection. Such forms should not prevent the composition or formulationfrom reaching a target cell (Le., a cell to which the negatively chargednucleic acid is desirable for delivery). For example, compositionsinjected into the blood stream should be soluble. Other factors areknown in the art, and include considerations such as toxicity and formsthat prevent the composition or formulation from exerting its effect. Asused herein, pharmaceutical formulations include formulations for humanand veterinary use. Non-limiting examples of agents suitable forformulation with the nucleic acid molecules of the instant inventioninclude: Lipid Nanoparticles (see for example Semple et al., 2010, NatBiotechnol., Feb; 28(2):172-6.); P-glycoprotein inhibitors (such asPiuronic P85); biodegradable polymers, such as poly(DL-lactide-coglycolide) microspheres for sustained release delivery(Emerich, D F et al, 1999, Cell Transplant, 8, 47-58); and loadednanoparticles, such as those made of polybutylcyanoacrylate. Othernon-limiting examples of delivery strategies for the nucleic acidmolecules of the instant invention include material described in Boadoet al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBSLett., 421, 280-284: Pardridge et al., 1995, PNAS USA., 92. 5592-5596:Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada etal., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et (W., 1999,PNAS USA., 96, 7053-7058. A “pharmaceutically acceptable composition” or“pharmaceutically acceptable formulation” can refer to a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant invention to the physical location mostsuitable for their desired activity.

The phrase “cytotoxicicytostatic agents” refer to compounds that causecell death or inhibit cell proliferation primarily by interferingdirectly with the cell's functioning or inhibit or interfere with cellmytosis, including alkylating agents, tumor necrosis factors,intercalators, hypoxia acuvatable compounds, microtubuleinhibitors/microWbule-stabilizing agents, inhibitors of mitotickinesins, inhibitors of histone deacetylase, inhibitors of kinasesinvolved in mitotic progression, antimetabolites; biological responsemodifiers; hormonal/anti-hormonal therapeutic agents, hematopoieticgrowth factors, monoclonal antibody targeted therapeutic agents,topoisomerase inhibitors, proteasoine inhibitors and ubiquitin ligaseinhibitors.

The phrase “estrogen receptor modulators” refers to compounds thatinterfere with or inhibit the binding of estrogen to the receptor,regardless of mechanism.

The term “gene” or “target gene” as used herein refers to their meaningas is generally accepted in the art. The terms generally refer a nucleicacid DNA or RNA) sequence that comprises partial length or entire lengthcoding sequences necessary for the production of a polypeptide. Thetarget gene can also include the UTR or non-coding region of the nucleicacid sequence. A gene or target gene can also encode a functional RNA(fRNA) or non-coding RNA (ncRNA), such as small temporal RNA (stRNA),micro RNA (miRNA), small nuclear RNA (snRNA), short interfering RNA(siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA(tRNA) and precursor RNAs thereof. Such non-coding RNAs can serve astarget nucleic acid molecules for siNA mediated RNA interference inmodulating the activity of fRNA or ncRNA involved in functional orregulatory cellular processes. Aberrant fRNA or ncRNA activity leadingto disease can therefore be modulated by siNA molecules of theinvention. siNA molecules targeting fRNA and ncRNA can also be used tomanipulate or alter the genotype or phenotype of a subject, organism orcell, by intervening in cellular processes such as genetic imprinting,transcription, translation, or nucleic acid processing (e.g.,transamination, methylation etc.). The target gene can be a gene derivedfrom a cell, an endogenous gene, a transgene, or exogenous genes such asgenes of a pathogen, for example a virus, which is present in the cellafter infection thereof. The cell containing the target gene can bederived from or contained in any organism, for example a plant, animal,protozoan, virus, bacterium, or fungus. Non-limiting examples of plantsinclude monocots, dicots, or gymnosperm's. Non-limiting examples ofanimals include vertebrates or invertebrates. Non-limiting examples offungi include molds or yeasts. For a review, see for example Snyder andGerstein, 2003, Science, 300, 258-260. In certain embodiments, genetargets contemplated herein are also referred to herein generally as“target” sequences (including the target sequences listed by GenBankAccession numbers in U.S. Ser. No. 60/363,124, incorporated by referenceherein).

The phrase “HMG-CoA reductase inhibitors” refers to inhibitors of3-hydroxy-3-methylglutaryl-CoA reductase. The term HMG-CoA reductaseinhibitor as used herein includes all pharmaceutically acceptablelactone and open-acid forms (i.e., where the lactone ring is opened toform the free acid) as well as salt and ester forms of compounds thathave HMG-CoA reductase inhibitory activity, and therefore the use ofsuch salts, esters, open-acid and lactone forms is included within thescope of this invention.

The phrase “highly conserved sequence region” refers to a nucleotidesequence of one. or more regions in a target gene that does not varysignificantly from one generation to the other or from one biologicalsystem to the other.

The phrase “homologous sequence” as used herein refers to its meaning asis generally accepted in the art. The term generally refers a nucleotidesequence that is shared by one or more polynucleotide sequences, such asgenes, gene transcripts and/or non-coding polynucleotides. For example,a homologous sequence can be a nucleotide sequence that is shared by twoor more genes encoding related but different proteins, such as differentmembers of a gene family, different protein epitopes, different proteinisoforms or completely divergent genes. A homologous sequence can be anucleotide sequence that is shared by two or more non-codingpolynucleotides, such as noncoding DNA or RNA, regulatory sequences,introns, and sites of transcriptional control or regulation. Homologoussequences can also include sequence regions shared by more than onepolynucleotide sequence. The term “perfect homology” (or “perfectlyhomologous”) as used herein refers to complete (100%) homology or“identity” between a reference sequence and a subject nucleic acidsequence. Homology does not need to be perfect identity (100%), however,as partially homologous sequences are also contemplated by and withinthe scope of the instant invention (e.g., at least 95%, 94%, 93%, 92%,91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).Percent homology is the number of matching nucleotides between twosequences divided by the total length being compared, multiplied by 100.

The phrase “improved RNAi activity” refers to an increase in RNAiactivity measured in vitro and/or in vivo, where the RNAi activity is areflection of both the ability of the siNA to mediate RNAi and thestability of the siNAs of the invention. In this invention, the productof these activities can be increased in vitro and/or in vivo compared toan all RNA siNA or an siNA containing a plurality of ribonucleotides. Insome cases, the activity or stability of the siNA molecule can bedecreased (i.e., less than ten-fold), but the overall activity of thesiNA molecule is enhanced in vitro and/or in vivo.

The term “including” (and any form thereof, such as “includes” and“include”), “comprising” (and any form thereof, such as “has” or “have”)or “containing” (and any form thereof such as “contains” or “contain”)are inclusive and open-ended and do not exclude additional, unrecitedelements or method steps.

The terms “inhibit,” “down-regulate,” or “reduce” as used herein refersto its meaning as is generally accepted in the art. With reference toexemplary nucleic acid molecules of the invention, be term generallyrefers the reduction in the expression of the gene, or level of RNAmolecules or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits, below that observed in the absence of the nucleic acidmolecules (e.g., siNA) of the invention. Down-regulation can also beassociated with post-transcriptional silencing, such as, RNAi mediatedcleavage or by alteration in DNA methylation patterns or DNA chromatinstructure. Inhibition, down-regulation or reduction with an siNAmolecule can be in reference to an inactive molecule, an attenuatedmolecule, an siNA molecule with a scrambled sequence, or an siNAmolecule with mismatches or alternatively, it can be in reference to thesystem in the absence of the nucleic acid.

The phrase “inhibitors of cell proliferation and survival signalingpathway” refers to pharmaceutical agents that inhibit cell surfacereceptors and signal transduction cascades downstream of those surfacereceptors.

The term “integrin blockers” refers to compounds which selectivelyantagonize, inhibit or counteract binding of a physiological ligand tothe αωβ₃ integrin, to compounds which selectively antagonize, inhibit orcounteract binding of a physiological ligand to the αωβ₅ integrin, tocompounds which antagonize, inhibit or counteract binding of aphysiological ligand to both the αωβ₃ integrin and the αωβ₅ integrin,and to compounds which antagonize, inhibit or counteract the activity ofthe particular integrin(s) expressed on capillary endothelial cells. Theterm also refers to antagonists of the αωβ₆ αωβ₈ α₁β₁ α₂β α₅β₁ α₆β₁ andα₆β₄ integrins. The term also refers to antagonists of any combinationof αωβ₃, αωβ₅, αωβ₆ αωβ₈ α₁β₁ α₂β₁ α₅β₁ and α₆β₄ integrins.

The terms “intermittent” or “intermittently” as used herein refers toits meaning as is generally accepted in the art. The term generallyrefers to periodic stopping and starting at either regular or irregularintervals.

The terms “internucleoside linkage” or “internucleoside linker” or“internucleotide linkage” or “internucleotide linker” are used hereininterchangeably and refer to any linker or linkage between twonucleoside units, as is known in the art, including, for example, butnot limitation, phosphate, analogs of phosphate, phosphonate,guanidinium, hydroxylamine, hydroxylhydrazinyl, amide, carbamate, alkyl,and substituted alkyl. linkages. The internucleoside linkages constitutethe backbone of a nucleic acid molecule.

The term “ligand” or refers to such compounds and compositions as aregenerally known in the art. Non-limiting examples of such ligands aredescribed herein including in the documents specifically incorporated byreference herein. A siNA. molecule of the invention can be formulated oradministered with any covalently linked ligand as described herein orotherwise known in the alt.

The term “lipid nanoparticle” or “LNP” refers to lipid-basedcompositions and formulations as are generally known in the art.Non-limiting examples of such LNPs are described herein including in thedocuments specifically incorporated by reference herein. A siNA moleculeof the invention can be formulated or administered with any LNP asdescribed herein or otherwise known in the art.

The terms “mammalian” or “mammal” as used herein refers to its meaningas is generally accepted in the art. The term generally refers to anywarm blooded vertebrate species, such as a human, mouse, rat, dog, cat,hamster, guinea pig, rabbit, livestock, and the like.

The phrase “metered dose inhaler” or MDI refers to a unit comprising acan, a secured cap covering the can and a formulation metering valvesituated in the cap. MDI systems includes a suitable channeling device.Suitable channeling devices comprise for example, a valve actuator and acylindrical or cone-like passage through which medicament can bedelivered from the filled canister via the metering valve to the nose ormouth of a patient such as a mouthpiece actuator.

The term “microRNA” or “miRNA” as used herein refers to its meaning asis generally accepted in the art. The term generally refers to anendogenous short RNA molecule found in eukaryotes that is involved inRNA-based gene regulation. A representative set of known endogenousmiRNA species is described in the publicly available miRBase sequencedatabase as described in Griffith-Jones et al., Nucleic Acids Research,2004, 32:D109-D111 and Griffith-Jones et al., Nucleic Acids Research,2006. 34:D 140-D144, accessible on the World Wide Web at the WellcomeTrust Sanger Institute website. Each mature miRNA is partiallycomplementary to one or more messenger RNA (mRNA) molecules, which arealso called “miRNA targets,” thereby regulating the expression of genesassociated with the miRNA targets.

The term “modulate” as used herein refers to its meaning as is generallyaccepted in the art. With reference to exemplary nucleic acid moleculesof the invention, the term refers to when the expression of a gene, orlevel of one or more RNA molecules (coding or non-coding), or activityof one or more RNA molecules or proteins or protein subunits, isup-regulated or down-regulated, such that expression, level, or activityis greater than or less than that observed in the absence of themolecule that effects modulation. For example, the term “modulate” insome embodiments can refer to inhibition and in other embodiments canrefer to potentiation or up-regulation, e.g., of gene expression.

The phrase “modified nucleotide” as used herein refers to its meaning asis generally accepted in the art. The term generally refers anucleotide, which contains a modification in the chemical structure ofthe base, sugar and/or phosphate of the unmodified (or natural)nucleotide as is generally known in the art. Non-limiting examples ofmodified nucleotides are described herein and in U.S. application Ser.No. 12/064,014.

The phrase “NSAIDs that are selective COX-2 inhibitors” for purposesherein, refers to NSAIDs, which possess a specificity for inhibitingCOX-2 over COX-1 of at least 100 fold as measured by the ratio of IC₅₀for COX-2 over IC₅₀ for COX-1. evaluated by cell or microsomal assays.

The phrase “non-base paired” refers to nucleotides that are not. basepaired between the sense strand or sense region and the antisense strandor antisense region of an double-stranded siNA molecule; and can includefor example, but not limitation, mismatches, overhangs, single strandedloops, etc.

The term “non-nucleotide.” refers to any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, such as for example but not limitation abasic moietiesor alkyl chains. The group or compound is “abasic” in that it does notcontain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine and therefore lacks a nucleobase atthe 1′-position.

The term “nucleotide” is used as is generally recognized in the art.Nucleotides generally comprise a nucleobase, a sugar, and aninternucleoside linkage, e.g., a phosphate. The base can be a naturalbases (standard), modified bases, or a base analog, as are well known inthe art. Such bases are generally located at the 1′ position of anucleotide sugar moiety. Additionally, the nucleotides can be unmodifiedor modified at the sugar, internucleoside linkage, and/or base moiety,(also referred to interchangeably as nucleotide analogs, modifiednucleotides, non-natural nucleotides, non-standard nucleotides andothers; see, for example, U.S. application Ser. No. 12/064,014.

The term “overhang” as used herein refers to its meaning as is generallyaccepted in the art. With reference to exemplary double stranded nucleicacid molecules, the term generally refers to the terminal portion of anucleotide sequence that is not base paired between the two strands of adouble-stranded nucleic acid molecule (see for example, FIGS. 5A-C).Overhangs, when present, are typically at the 3′-end of one or bothstrands in a siNA duplex.

The term “parenteral” as used herein refers to its meaning as isgenerally accepted in the art, The term generally refers methods ortechniques of administering a molecule, drug, agent, or compound in amanner other than through the digestive tract, and includesepicutaneous, subcutaneous, intravascular (e.g., intravenous),intramuscular, or intrathecal injection or infusion techniques and thelike.

The phrase “pathway target” refers to any target involved in pathways ofgene expression or activity. For example, any given target can haverelated pathway targets that can include upstream, downstream, ormodifier genes in a biologic pathway. These pathway target genes canprovide additive or synergistic effects in the treatment of diseases,conditions, and traits herein.

The term “phosphorothioate” refers to an internucleotide phosphatelinkage comprising one or more sulfur atoms in place of an oxygen atom.Hence, the term phosphorothioate refers to both phosphorothioate andphosphorodithioate internucleotide linkages.

The term “polymer” refers to polymeric compounds, compositions andformulations as are generally known in the art. Non-limiting examples ofsuch polymers, including polymeric delivery systems are described hereinincluding in the documents specifically incorporated by referenceherein. A siNA molecule of the invention can be formulated oradministered with any polymer as described herein or otherwise known inthe art.

The term “position 1” refers to the position of the first nucleotide atthe end of a strand, e.g., antisense strand. All positions referred toherein are the positions of a nucleotide counting from the end of astrand, for example, positions 1-3 from the 5′ end of the antisensestrand, refer to the three nucleotides at positions 1, 2, and 3 countingfrom the 5′ end of the antisense strand.

The term “ribonucleotide” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to a nucleotidewith a hydroxyl group at the 2′ position of a β-D-ribofuranose moiety.

The term “RNA” as used herein refers to its generally accepted meaningin the art. Generally, the term RNA refers to a molecule comprising atleast one ribofuranoside moiety. The term can include double-strandedRNA, single-stranded RNA, isolated RNA such as partially purified RNA,essentially pure RNA, synthetic RNA, recombinantly produced RNA, as wellas altered RNA that differs from naturally occurring RNA by theaddition, deletion, substitution and/or alteration of one or morenucleotides. Such alterations can include addition of non-nucleotidematerial, such as to the ends) of the siNA or internally, for example atone or more nucleotides of the RNA. Nucleotides in the RNA molecules ofthe instant invention can also comprise non-standard nucleotides, suchas non-naturally occurring nucleotides or chemically synthesizednucleotides or deoxynucleotides. These altered RNAs can be referred toas analogs or analogs of naturally-occurring RNA.

The phrase “RNA interference” or term “RNAi” refer to the biologicalprocess of inhibiting or down regulating gene expression in a cell, asis generally known in the art, and which is mediated by shortinterfering nucleic acid molecules, see for example Zamore and Haley,2005, Science, 309, 1519-1524; Vaughn end Martienssen, 2005, Science,309, 1525-1526 Zamore et al., 2000, Cell, 101, 25-33; Bass, 2001,Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; andKreutzer et al., International PCT Publication No. WO 00/44895;Zernicka-Goetz et al., International PCT Publication No. WO 01/36646;Fire, International PCT Publication No. WO 99/32619; Plaetinck et al.,International PCT Publication No. WO 00/01846; Mello and Fire,International PCT Publication No. WO 01/29058; Deschamps-Depaillette,International PCT Publication No. WO 99/07409; and Li et InternationalPCT Publication No. WO 00/44914; Allshire, 2002, Science, 297,1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002,Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002,RNA, 8, 842-850; Reinhart et 2002, Gene & Dev., 16, 1616-1626; andReinhart & Bartel, 2002, Science, 297, 1831). Additionally, the termRNAi is meant to be equivalent to other terms used to describe sequencespecific RNA interference, such as post transcriptional gene silencing,translational inhibition, transcriptional inhibition, or epigenetics.For example, siNA molecules of the invention can be used toepigenetically silence genes at either the post-transcriptional level orthe pre-transcriptional level. In a non-limiting example, epigeneticmodulation of gene expression by siNA molecules of the invention canresult from siNA mediated modification of chromatin structure orarethylation patterns to alter gene expression (see, for example, Verdeet al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004Science,303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al.,2002, Science, 297, 1833-1837: Jenuwein, 2002, Science, 297, 2215-2218;and Hall et al., 2002, Science, 297, 2232-2237′). In anothernon-limiting example, modulation of gene expression by siNA molecules ofthe invention can result from siNA mediated cleavage of RNA (eithercoding or non-coding RNA) via RISC, or via translational inhibition, asis known in the art or modulation can result from transcriptionalinhibition (see for example Janowski et al., 2005, Nature ChemicalBiology, 1, 216-222).

The phrase “RNAi inhibitor” refers to any molecule that can downregulate, reduce or inhibit RNA interference function or activity in acell or organism. An RNAi inhibitor can down regulate, reduce or inhibitRNAi (e.g., RNAi mediated cleavage of a target polynucleotide,translational inhibition, or transcriptional silencing) by interactionwith or interfering with the function of any component of the RNAipathway, including protein components such as RISC, or nucleic acidcomponents such as miRNAs or siRNAs. A RNAi inhibitor can be an siNAmolecule, an antisense molecule, an aptamer, or a small molecule thatinteracts with or interferes with the function of RISC, a miRNA, or ansiRNA or any other component of the RNAi pathway in a cell or organism.By inhibiting RNAi RNAi mediated cleavage of a target polynucleotide,translational inhibition, or transcriptional silencing), a RNAiinhibitor of the invention can be used to modulate (e.g., up-regulate ordown regulate) the expression of a target gene.

The phrase “sense region” as used herein refers to its meaning as isgenerally accepted in the art. With reference to exemplary nucleic acidmolecules of the invention, the term refers to a nucleotide sequence ofan siNA molecule having complementarity to an antisense region of thesiNA molecule. In addition, the sense region of an siNA molecule cancomprise a nucleic acid sequence having homology or sequence identitywith a target nucleic acid sequence. In one embodiment, the sense regionof the siNA molecule is also referred to as the sense strand orpassenger strand.

The phrases “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically modified shortinterfering nucleic acid molecule” refer to any nucleic acid moleculecapable of inhibiting or down regulating gene expression or viralreplication by mediating RNA interference (“RNAi”) or gene silencing ina sequence-specific manner. These terms can refer to both individualnucleic acid molecules, a plurality of such nucleic acid molecules, orpools of such nucleic acid molecules. The siNA can be a double-strandednucleic acid molecule comprising self-complementary sense and antisensestrands, wherein the antisense strand comprises a nucleotide sequencethat is complementary to a nucleotide sequence in a target nucleic acidmolecule or a portion thereof and the sense strand comprises anucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof. The siNA can be a polynucleotide with a duplex,asymmetric duplex, hairpin or asymmetric hairpin secondary structure,having self-complementary sense and antisense regions, wherein theantisense region comprises a nucleotide sequence that is complementaryto a nucleotide sequence in a separate target nucleic acid molecule or aportion thereof and the sense region comprises a nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof.The siNA can be a circular single-stranded polynucleotide having two ormore loop structures and a stem comprising self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to a nucleotide sequence in a targetnucleic acid molecule or a portion thereof and the sense regioncomprises a nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof, and wherein the circular polynucleotidecan be processed either in vivo or in vitro to generate an active siNAmolecule capable of mediating RNAi. The siNA can also comprise asingle-stranded polynucleotide having a nucleotide sequencecomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof (for example, where such siNA molecule does notrequire the presence within the siNA molecule of a nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof),wherein the single-stranded polynucleotide can further comprise aterminal phosphate group, such as a 5′-phosphate (see for example,Martinez et al., 2002, Cell, 110, 563-574 and Schwarz et al., 2002,Molecular Cell, 10, 537-568). or 5′,3′-diphosphate.

The term “subject” as used herein refers to its meaning as is generallyaccepted in the art. The term generally refers an organism to which thenucleic acid molecules of the invention can be administered. A subjectcan be a mammal or mammalian cells, including a human or human cells.The term also refers to an organism, which is a donor or recipient ofexplanted cells or the cells themselves.

The phrase “systemic administration” as used herein refers to itsmeaning as is generally accepted in the art. The term generally refersin vivo systemic absorption or accumulation of drugs in the blood streamfollowed by distribution throughout the entire body.

The term “target” as used herein refers, to any protein, peptide, orpolypeptide, such as encoded by any gene in the GenBank database,including those described herein and/or in U.S. Provisional PatentApplication No. 60/363,124, U.S. application Ser. No. 10/923,536 and/orPCT/US03/05028, all of which are incorporated herein by reference forpurposes of identifying such targets. The term “target” also refers toone or more genes, nucleic acid sequences, or target polynucleotidesequences encoding any target protein, peptide, or polypeptide, such asproteins, peptides, or polypeptides encoded by the genes in the Genebankdatabase or sequences having GenBank Accession Nos. shown herein and/orin U.S. Provisional Patent Application No. 60/363,124, U.S. applicationSer. No. 10/923,536 and: or PCT/US03/05028, all of which areincorporated herein by reference for purposes of identify such targets.The target of interest can include target polynucleotide sequences, suchas target DNA or target RNA. The term “target” is also meant to includeother sequences, such as differing isoforms, mutant target genes, splicevariants of target polynucleotides, target polymorphisms, and non-coding(e.g., ncRNA, miRNA, stRNA) or other regulatory polynucleotide sequencesas described herein. Therefore, in various embodiments of the invention,a double stranded nucleic acid molecule of the invention (e.g., siNA)having complementarity to a target RNA can be used to inhibit or downregulate miRNA or other ncRNA activity. In one embodiment, inhibition ofmiRNA or ncRNA activity can be used to down regulate or inhibit geneexpression (e.g., gene targets described herein or otherwise known inthe art) that is dependent on miRNA or ncRNA activity. In anotherembodiment, inhibition of miRNA or ncRNA activity by double strandednucleic acid molecules of the invention (e.g. siNA) havingcomplementarity to the miRNA or ncRNA can be used to up regulate orpromote target gene expression (e.g., gene targets described herein orotherwise known in the art) where the expression of such genes is downregulated, suppressed, or silenced by the miRNA or ncRNA. Suchup-regulation of gene expression can be used to treat diseases andconditions associated with a loss of function or haploinsufficiency asare generally known in the art.

The phrase “target site” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to a sequencewithin a target nucleic acid molecule, (e.g., RNA) that is “targeted”,e,g., for cleavage mediated by an siNA construct, which containssequences within its antisense region that are complementary to thetarget sequence.

The phrase “therapeutically effective amount” as used herein refers toits meaning as is generally accepted in the art. The term generallyrefers to the amount of the compound or composition that will elicit thebiological or medical response of a cell, tissue, system, animal orhuman that is be sought by the researcher, veterinarian, medical doctoror other clinician. For example, if a given clinical treatment isconsidered effective when there is at least a 25% reduction in ameasurable parameter associated with a disease or disorder, atherapeutically effective amount of a drug for the treatment of thatdisease or disorder is that amount necessary to effect at least a 25%reduction in that parameter.

The phrase “universal base” as used herein refers to its meaning as isgenerally accepted in the art. The term universal base generally refersto nucleotide base analogs that form base pairs with each of the naturalDNA/RNA bases with little or no discrimination between them.Non-limiting examples of universal bases include C-phenyl, C-naphthyland other aromatic derivatives, inosine, azole carboxamides, andnitroazole derivatives such as 3-nitropyrrole, 4-nitroindole,5-nitroindole, and 6-nitroindole as known in the art (see for example,Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

The term “up-regulate” as used herein refers to its meaning as isgenerally accepted in the art. With reference to exemplary nucleic acidmolecules of the invention, the term refers to an increase in theexpression of a gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more RNAs, proteins or protein subunits, above that observedin the absence of the nucleic acid molecules siNA) of the invention. Incertain instances, up-regulation or promotion of gene expression with ansiNA molecule is above that level observed in the presence of aninactive or attenuated molecule. In other instances, up-regulation orpromotion of gene expression with siNA molecules is above that levelobserved in the presence of, for example, an siNA molecule withscrambled sequence or with mismatches. In still other instances,up-regulation or promotion of gene expression with a nucleic acidmolecule of the instant invention is greater in the presence of thenucleic acid molecule than in its absence. In some instances,up-regulation or promotion of gene expression is associated withinhibition of RNA mediated gene silencing, such as RNAi mediatedcleavage or silencing of a coding or non-coding RNA target that downregulates, inhibits, or silences the expression of the gene of interestto be up-regulated. The down regulation of gene expression can, forexample, be induced by a coding RNA or its encoded protein, such asthrough negative feedback or antagonistic effects. The down regulationof gene expression can, for example, be induced by a non-coding RNAhaving regulatory control over a gene of interest, for example bysilencing expression of the gene via translational inhibition, chromatinstructure, methylation, RISC mediated RNA cleavage, or translationalinhibition. As such, inhibition or down regulation of targets that downregulate, suppress, or silence a gene of interest can be used toup-regulate expression of the gene of interest toward therapeutic use.

The term “vector” as used herein refers to its meaning as is generallyaccepted in the art. The term vector generally refers to any nucleicacid- and/or viral-based expression system or technique used to deliverone or more nucleic acid molecules,

B. siNA Molecules of the Invention

The present invention provides compositions and methods comprising siNAshaving target specificity that can be used to treat diseases andconditions herein or otherwise known in the art that are associated withgene expression. In particular aspects and embodiments of the invention,the nucleic acid molecules of the invention comprise at least a 15nucleotide sequence of the a target sequence, and/or comprises anucleotide sequence of at least 15 nucleotides complimentary to thetarget sequence). The siNAs can be provided in several forms. Forexample, the siNA can be isolated as one or more siNA compounds, or itmay be in the form of a transcriptional cassette in a DNA plasmid. ThesiNA may also be chemically synthesized and can include modifications asshown, for example, but not limitation, in Table 1 and Table 8. ThesiNAs can be administered alone or co-administered with other siNAmolecules or with conventional agents that treat a gene related diseaseor condition as described herein or otherwise known in the art.

The siNA molecules of the invention can be used to mediate genesilencing via interaction with RNA transcripts or alternately byinteraction with particular gene sequences, wherein such interactionresults in modulation of gene silencing either at the transcriptionallevel or post-transcriptional level such as, for example, but notlimited to, RNAi or through cellular processes that modulate thechromatin structure or methylation patterns of the target and preventtranscription of the target gene, with the nucleotide sequence of thetarget thereby mediating silencing. More specifically, the target is anyGenBank reference sequence as is presently known in the art.

In one aspect, the invention provides short interfering nucleic acid(siNA) molecules for inhibiting the expression of the target gene in acell or mammal. The siNA can be single-stranded or double-stranded. Whendouble-stranded, the siNA comprising a sense and an antisense stand. Theantisense strand is complementary to at least a part of an mRNA formedin the expression of the HBV gene. The sense strand comprises a regionthat is complementary to the antisense strand. One or more of thenucleotides of the siNAs of the invention are optionally modified.

The double stranded RNA molecules of the invention can comprise twodistinct and separate strands that can be symmetric or asymmetric andare complementary, i.e., two single-stranded RNA molecules, or cancomprise one single-stranded molecule in which two complementaryportions, e.g., a sense region and an antisense region, are base-paired,and are covalently linked by one or more single-stranded “hairpin” areas(i.e. loops) resulting in, for example, a single-stranded short-hairpinpolynucleotide or a circular single-stranded polynucleotide.

The linker can be polynucleotide linker or a non-nucleotide linker. Insome embodiments, the linker is a non-nucleotide linker. In someembodiments, a hairpin or circular siNA molecule of the inventioncontains one or more loop motifs, wherein at least one of the loopportions of the siNA molecule is biodegradable. For example, asingle-stranded hairpin siNA molecule of the invention is designed suchthat degradation of the loop portion of the. siNA molecule in vivo cangenerate a double-stranded siNA molecule with 3′-terminal overhangs,such as 3′-terminal nucleotide overhangs comprising 1, 2, 3 or 4nucleotides. Or alternatively, a circular siNA molecule of the inventionis designed such that degradation of the loop portions of the siNAmolecule in vivo can generate a double-stranded siNA molecule with3′-terminal overhangs, such as 3′-terminal nucleotide overhangscomprising about 2 nucleotides.

In some embodiments, siNA molecules of the invention have perfectcomplementarity between the sense strand or sense region and theantisense strand or antisense region of the siNA molecule. In other orthe same embodiments, the antisense strand of the siNA molecules of theinvention are perfectly complementary to a corresponding target nucleic.acid molecule.

In yet other embodiments, siNA molecules of the invention have partialcomplementarity , less than 100% complementarity) between the sensestrand or sense region and the antisense strand or antisense region ofthe siNA molecule or between the antisense strand or antisense region ofthe siNA molecule and a corresponding target nucleic acid molecule.Thus, in some embodiments, the double-stranded nucleic acid molecules ofthe invention, have between about 15 to about 30 (e.g., about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotidesin one strand that are complementary to the nucleotides of the otherstrand. In other embodiments, the molecules have between about 15 toabout 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 2.2, 23, 24, 25, 26,27, 28, 29, or 30) nucleotides in the sense region that arecomplementary to the nucleotides of the antisense region. of thedouble-stranded nucleic acid molecule. In certain embodiments, thedouble-stranded nucleic acid molecules of the invention have betweenabout 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30) nucleotides in the antisense strand thatare complementary to a nucleotide sequence of its corresponding targetnucleic acid molecule.

In other embodiments, the siNA molecule can contain one or morenucleotide deletions, substitutions, mismatches and/or additions;provided, however, that the siNA molecule maintains its activity, forexample, to mediate RNAi. In a non-limiting example, the deletion,substitution, mismatch and/or addition can result in a loop or bulge, oralternately wobble or other alternative (non Watson-Crick) base pair.Thus, in some embodiments, for example, the double-stranded nucleic acidmolecules of the invention, have 1 or more 1, 2, 3, 4, 5, or 6)nucleotides, in one strand or region that are mismatches ornon-base-paired with the other strand or region. In other embodiments,the double-stranded nucleic acid molecules of the invention, have 1 ormore (e.g., 1, 2, 3, 4, 5, or 6) nucleotides in each strand or regionthat are mismatches or non-base-paired with the other strand or region.In a preferred embodiment, the siNA of the invention contains no morethan 3 mismatches. If the antisense strand of the siNA containsmismatches to a target sequence, it is preferable that the area ofmismatch not be located in the center of the region of complementarity.

In other embodiments, the siNA molecule can contain one or morenucleotide deletions, substitutions, mismatches and/or additions to asequence provided herein, however, that the siNA molecule maintains itsactivity, for example, to mediate RNAi. In a non-limiting example, thedeletion, substitution, mismatch and/or addition can result in a loop orbulge, or alternately a wobble or other alternative (non Watson-Crick)base pair.

The invention also comprises double-stranded nucleic acid (siNA)molecules as otherwise described hereinabove in which the first strandand second strand are complementary to each other and wherein at leastone strand is hybridisable to the polynucleotide sequence of a targetsequence under conditions of high stringency, and wherein any of thenucleotides is unmodified or chemically modified.

Hybridization techniques are well known to the skilled artisan (see forinstance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989)). Preferred stringent hybridization conditions include overnightincubation at 42° C. in a solution comprising: 50% formamide, 5× SSC(1.50 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 microgram/mldenatured, sheared salmon sperm DNA; followed by washing the filters in0.1× SSC at about 65° C.

In some embodiments, the first strand has about 15, 16, 17, 18, 19, 20or 21 nucleotides that are complementary to the nucleotides of the otherstrand and at least one strand hybridisable to target sequence such as agene in the GenBank database. In a more preferred embodiment, the firststrand has about 15, 16, 17, 18, 19, 20 or 21 nucleotides that arecomplementary to the nucleotides of the other strand and at least onestrand is hybridisable at least one strand is hybridisable to thecomplement of a target sequence under conditions of high stringency; andwherein any of the nucleotides is unmodified or chemically modifiedexcept that positions 1-3 of the 5′ end of the antisense strand aremodified. .

In certain embodiments, the siNA molecules of the invention compriseoverhangs of about 1 to about 4 (e.g., about 1, 2, 3 or 4) nucleotides.The nucleotides in the overhangs can be the same or differentnucleotides. In some embodiments, the overhangs occur at the 3′-end atone or both strands of the double-stranded nucleic acid molecule. Forexample, a double-stranded nucleic acid molecule of the invention cancomprise a nucleotide or non-nucleotide overhang at the 3′-end of theantisense strand/region, the 3′-end of the sense strand/region, or boththe antisense strand/region and the sense strand/region of thedouble-stranded nucleic acid molecule.

In some embodiments, the nucleotides comprising the overhang portion ofan siNA molecule of the invention comprise sequences based on the targetpolynucleotide sequence in which nucleotides comprising the overhangportion of the antisense strand/region of an siNA molecule of theinvention can be complementary to nucleotides in the targetpolynucleotide sequence and/or nucleotides comprising the overhangportion of the sense strand/region of an siNA molecule of the inventioncan comprise the nucleotides in the target polynucleotide sequence.Thus, in some embodiments, the overhang comprises a two nucleotideoverhang that is complementary to a portion of the target polynucleotidesequence. In other embodiments, however, the overhang comprises a twonucleotide overhang that is not complementary to a portion of the targetpolynucleotide sequence. In certain embodiments, the overhang comprisesa 3′UU overhang that is not complementary to a portion of the targetpolynucleotide sequence. In other embodiments, the overhang comprises aULT overhang at the 3′ end of the antisense strand and a TT overhang atthe 3′ end of the sense strand. In other embodiments, the overhangcomprises nucleotides as described in the examples, Tables, and Figuresherein.

In any of the embodiments of the siNA molecules described herein having3′-terminal nucleotide overhangs, the overhangs are optionallychemically modified at one or more nucleic acid sugar, base, or backbonepositions. Representative, but not limiting examples of modifiednucleotides in the overhang portion of a double-stranded nucleic acid(siNA) molecule of the invention include: 2′-O-alkyl (e.g 2′-O-methyl),2′-deoxy, 2′-deoxy-2′-fluoro, 2′-deoxy-2′-fluoroarabino (FANA), 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy, universal base, acyclic, or 5—C-methylnucleotides. In more preferred embodiments, the overhang nucleotides areeach independently, a 2′-O-alkyl nucleotide, a 2′-O-methyl nucleotide, a2′-dexoy-2-fluoro nucleotide, or a 2′-deoxy ribonucleotide. In someinstances the overhang nucleotides are linked by a one or morephosphorothioate linkages.

In yet other embodiments, siNA molecules of the invention compriseduplex nucleic acid molecules with blunt ends (i.e without nucleotideoverhangs), where both ends are blunt, or alternatively, where one ofthe ends is blunt. In some embodiments, the siNA molecules of theinvention can comprises one blunt end, for example wherein the 5′-end ofthe antisense strand and the 3′-end of the sense strand do not have anyoverhanging nucleotides, In another example, the siNA molecule comprisesone blunt end, for example wherein the 3′-end of the antisense strandand the 5′-end of the sense strand do not have any overhangingnucleotides. In other embodiments, siNA molecules of the inventioncomprise two blunt ends, for example wherein the 3″-end of the antisensestrand and the 5′-end of the sense strand as well as the 5′-end of theantisense strand and 3′-end of the sense strand do not have anyoverhanging nucleotides.

In any of the embodiments or aspects of the siNA molecules of theinvention, the sense strand and/or the antisense strand can further havea cap, such as described herein or as known in the art, at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends of the sense strand and/orantisense strand. Or as in the case of a hairpin siNA molecule, the capcan be at either one or both of the terminal nucleotides of thepolynucleotide. In some embodiments, the cap is at one of both of theends of the sense strand of a double-stranded siNA molecule. In otherembodiments, the cap is at the 3′-end of antisense (guide) strand. Inpreferred embodiments, the caps are at the 3′-end of the sense strandand the 5′-end of the sense strand.

Representative, but non-limiting examples of such terminal caps includean inverted abasic nucleotide, an inverted deoxy abasic nucleotide, aninverted nucleotide moiety, a group shown in FIG. 6 or FIG. 10, aglyceryl modification, an alkyl or cycloalkyl group, a heterocycle, orany other cap as is generally known in the art.

Any of the embodiments of the siNA molecules of the invention can have a5′ phosphate termini. In some embodiments, the siNA molecules lackterminal phosphates.

Any siNA molecule or construct of the invention can comprise one or morechemical modifications. Modifications can be used to improve in vitro orin vivo characteristics such as stability, activity, toxicity, immuneresponse (e.g., prevent stimulation of an interferon response, aninflammatory or pro-inflammatory cytokine response, or a Toll-likeReceptor (TIF) response), and/or bioavailability,

Applicants describe herein chemically modified siNA molecules withimproved RNAi activity and/or stability compared to correspondingunmodified siNA molecules. Various chemically modified siNA motifsdisclosed herein provide the capacity to maintain RNAi activity that issubstantially similar to unmodified or minimally modified active siRNA(see for example Elbashir et al.,: 2001, EMBO J., 20:68′77-6888) whileat the same time providing nuclease resistance and pharmacokineticproperties suitable for use in therapeutic applications.

In various embodiments, the siNA molecules of the invention comprisemodifications wherein any (e.g., one or more or all) nucleotides presentin the sense and/or antisense strand are modified nucleotides (e.g.,wherein one nucleotide is modified, some nucleotides (i.e., plurality ormore than one) are modified, or all nucleotides are modifiednucleotides. in some embodiments, the siNA molecules of the inventionare partially modified (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29.30, 31, 32., 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 nucleotides aremodified) with chemical modifications. In some embodiments, an siNAmolecule of the invention comprises at least about 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, or 60 nucleotides that are modified nucleotides. In otherembodiments, the siNA molecules of the invention are completely modified(e.g., 100% modified) with chemical modifications, i.e., the siNAmolecule does not contain any ribonucleotides. In some of embodiments,one or more of the nucleotides in the sense strand of the siNA moleculesof the invention are modified. In the same or other embodiments, one ormore of the nucleotides in the antisense strand of the siNA molecules ofthe invention are modified.

The chemical modification within a single siNA molecule can be the sameor different. In some embodiments, at least one strand has at least onechemical modification. In other embodiments, each strand has at leastone chemical modifications, which can be the same or different, such as,sugar, base, or backbone (i.e., internucleotide linkage) modifications.In other embodiments, siNA molecules of the invention contain at least:2, 3, 4, 5, or more different chemical modifications,

Non-limiting examples of chemical modifications that are suitable foruse in the present invention, are disclosed in U.S. patent applicationsSer. Nos. 10/444,853; 10/981,966; 12/064,014 and in references citedtherein and include sugar, base, and phosphate, non-nucleotidemodifications, and/or any combination thereof.

In certain specific embodiments of the invention, at least one modifiednucleotide is a 2′-deoxy-2-fluoro nucleotide, a 2′-deoxy nucleotide, a2′-O-alkyl (e.g., 2′-O-methyl) nucleotide, or a locked nucleic acid(LNA) nucleotide as: is generally recognized in the art.

In yet other embodiment of the invention, at least one nucleotide has aribo-like, Northern or A form helix configuration (see e.g., Saenger,Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984).Non-limiting examples of nucleotides having a Northern configurationinclude locked nucleic acid (LNA) nucleotides (e.g., 2′-O,4′-C-methylene-(D-ribofuranosyl) nucleotides); 2′-methoxyethoxy (MOE)nucleotides; 2′-methyl-thio-ethyl nucleotides, 2′-deoxy-2′-fluoronucleotides; 2′-deoxy-2′-chloro nucleotides; 2′-azido nucleotides;2′-O-trifluoromethyl nucleotides; 2′-O-ethyl-trifluoromethoxynucleotides; 2′-O-difluoromethoxy-ethoxy nucleotides; 4′-thionucleotides and 2′-t7-methyl nucleotides.

In various embodiments, a majority (e.g., greater than 50%) of thepyrimidine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification. In some of the same and/or otherembodiments, a majority (e.g., greater than 50%) of the purinenucleotides present in the double-stranded siNA molecule comprises asugar modification.

In some embodiments, the pyrimidine nucleotides in the antisense strandare 2′-O-methyl or 2′-deoxy-2′-fluoro pyrimidine nucleotides and thepurine nucleotides present in the. antisense strand are 2′-O-methylnucleotides or 2′-deoxy nucleotides. In other embodiments, thepyrimidine nucleotides in the sense strand are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in the sensestrand are 2′-O-methyl or 2′-deoxy purine nucleotides.

In certain embodiments of the invention, all the pyrimidine nucleotidesin the complementary region on the sense strand are 2′-deoxy-2′-fluoropyrimidine nucleotides. In certain embodiments, all of the pyrimidinenucleotides in the complementary region of the antisense strand are2′-deoxy-2′-fluoro pyrimidine nucleotides. In certain embodiments, allthe. purine nucleotides in the complementary region on the sense strandare 2′-deoxy purine nucleotides. In certain embodiments, all of thepurines in the complementary region on the antisense strand are2′-O-methyl purine nucleotides. In certain embodiments, all of thepyrimidine nucleotides in the complementary regions on the sense strandare 2′-deoxy-2′-fluoro pyrimidine nucleotides; all of the pyrimidinenucleotides in the complementary region of the antisense strand are2′-deoxy-2′-fluoro pyrimidine nucleotides; all the purine nucleotides inthe complementary region on the sense strand are 2′-deoxy purinenucleotides and all of the purines in the complementary region on theantisense strand are 2′-O-methyl purine nucleotides.

In some embodiments, at least 5 or more of the pyrimidine nucleotides inone or both stands are 2′-deoxy-2′-fluoro pyrimidine nucleotides. Insome embodiments, at least 5 or more of the pyrimidine nucleotides inone or both stands are 2′-O-methyl pyrimidine nucleotides. In someembodiments, at least 5 or more of the purine nucleotides in one or bothstands are 2′-deoxy-2′-fluoro purine nucleotides In some embodiments, atleast 5 or more of the purine nucleotides in one or both stands are2′-O-methyl purine nucleotides.

In some embodiments, at least 5 or more of the pyrimidine nucleotides inone or both strands are 2′-O-methyl nucleotides and at least 5 or moreof the purine nucleotides are 2′-deoxy-2-fluoro nucleotides. In someembodiments, at least 5, 6, 7, 8, 9, 10 or more of the pyrimidinenucleotides in one or both strands are 2′-O-methyl nucleotides and atleast 5, 6, 7, 8, 9, 10 or more of the purine nucleotides are2′-deoxy-2-fluoro nucleotides. In some embodiments, at least 5 or moreof the purine nucleotides in one or both stands are 2′-deoxy-2-fluoropurine nucleotides.

In certain embodiments, the purines and pyrimidines are differentiallymodified at the 2′-sugar position (i.e., at least one purine has adifferent modification from at least one pyrimidine in the same ordifferent strand at the 2′-sugar position). For example, in someinstances, at least 5 or more of the pyrimidine nucleotides in one orboth stands are 2′-deoxy-2′-fluoro pyrimidine nucleotides and at least 5or more purine nucleotides in one or both strands are 2′-O-methyl purinenucleotides. In other instances at least 5 or more of the pyrimidinenucleotides in one or both stands are 2′-O-methyl pyrimidine nucleotidesand at least 5 or more purine nucleotides in one or both strands are2′-deoxy-2′-fluoro purine nucleotides.

Further non-limiting examples of sense and antisense strands of suchsiNA molecules having various modifications are shown in FIGS. 2A-4 andTable 8.

Any of the above described modifications, or combinations thereof,including those in the references cited, can be applied to any of thesiNA molecules of the invention.

The modified siNA molecules of the invention can comprise modificationsat various locations within the siNA molecule. In some embodiments, thedouble-stranded siNA molecule of the invention comprises modifiednucleotides at internal base paired positions within the siNA duplex. Inother embodiments, a double-stranded siNA molecule of the inventioncomprises modified nucleotides at non-base paired or overhang regions ofthe siNA molecule. In yet other embodiments, a double-stranded siNAmolecule of the invention comprises modified nucleotides at terminalpositions of the siNA molecule. For example, such terminal regionsinclude the 3′-position and/or 5′-position of the sense and/or antisensestrand or region of the siNA molecule. Additionally, any of the modifiedsiNA molecules of the invention can have a modification in one or botholigonucleotide strands of the siNA duplex, for example in the sensestrand, the antisense strand, or both strands. Moreover, with regard tochemical modifications of the siNA molecules of the invention, eachstrand of the double-stranded siNA molecules of the invention can haveone or more chemical modifications, such that each strand comprises adifferent motif of chemical modifications.

In certain embodiments each strand of a double-stranded siNA molecule ofthe invention comprises a different motif of chemical modifications,such as any Stab modification chemistries described herein (see Table 8)or any combination thereof, i.e., different combinations of definedStabilization chemistry (Stab) sense and antisense strands. Further,non-limiting examples of modification schemes that could give rise todifferent motifs of modifications are shown in Table 8. Thestabilization chemistries referred to in Table 8 as Stab, can becombined in any combination of sense/antisense chemistries, such as Stab7/8, Stab 7/11, Stab 8/8, Stab 18/8, Stab 18/11, Stab 12/13, Stab 7/13,Stab 18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, Stab18/20, Stab 7/32, Stab 8/32, or Stab 18/32 or any other combination ofStabilization chemistries.

In any of the siNAs of the invention, one or more (for example 1, 2, 3,4 or 5) nucleotides at the 5′-end of the guide strand or guide region(also known as antisense strand or antisense region) of the siNAmolecule are ribonucleotides.

Any of the above described modifications, or combinations thereof,including those in the references cited, can be applied to any of theseembodiments.

In certain embodiments of the present invention, double-stranded siNAmolecules are provided that modulate the expression of a target gene viaRNA interference, wherein the molecule has a sense strand and anantisense strand and comprises structure represented by formula (A):

-   -   wherein, the upper strand is the sense strand and the lower        strand is the antisense strand of the double-stranded nucleic        acid molecule; wherein the antisense strand comprises a sequence        having at least 15 nucleotides that are complementary to a        target RNA sequence encoded by the target gene and the sense        strand comprises a sequence that is complementarity to the        antisense strand;    -   each N is independently a nucleotide which is unmodified or        chemically modified or is optionally a non-nucleotide;    -   each B is independently a terminal cap that is present or        absent;    -   (N) represents overhanging nucleotides, each of which is        independently unmodified or chemically modified;    -   [N] represents nucleotides at the 5′-terminus of the antisense        strand;    -   X1 and X2 are independently integers from 0 to 4;    -   X3 is an integer from 15 to 30;    -   X4 is an integer from 12 to 27; and    -   X5 is an integer from 1-6, provided that the sum of X4 and X5 is        an integer from 15-30.

Any of the above described modifications, or combinations thereof,including those in the references cited, can be applied to any of theseembodiments.

In certain embodiments, the nucleotides of the antisense strand sequencehaving at least 15 nucleotides complementary to a target sequence form acontiguous stretch of nucleotides.

In some embodiments, the siNA molecule of formula A can contain one ormore nucleotide deletions, substitutions, mismatches and/or additions tothe antisense strand sequence having at least 15 nucleotidescomplementary to a target sequence provided, however, that the siNAmolecule maintains its activity, for example, to mediate RNAL In anon-limiting example, the deletion, substitution, mismatch and/oraddition can result in a loop or bulge, or alternately a wobble or otheralternative (non Watson-Crick) base pair.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) of formula (A); wherein

-   -   one or more pyrimidine nucleotides in N_(X4) positions are        independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl        nucleotides, 2′-deoxy nucleotides, ribonucleotides, or any        combination thereof;    -   one or more purine nucleotides in N_(X4) positions are        independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl        nucleotides, 2′-deoxy nucleotides, ribonucleotides, or any        combination thereof;    -   one or more pyrimidine nucleotides in N_(X3) positions are        independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl        nucleotides, 2′-deoxy nucleotides, ribonucleotides, or any        combination thereof;    -   one or more purine nucleotides in N_(X3) positions are        independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl        nucleotides, 2′-deoxy nucleotides, ribonucleotides, or any        combination thereof; and

[N] position nucleotide(s) are ribonucleotides, deoxyribonucleotides,2′-O-alkyl nucleotides, 2′-halo nucleotides, or any combination thereofirrespective of purine or pyrimidine content.

In certain embodiments, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule of formula (A); wherein

-   -   5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides in N_(X4)        positions are 2′-deoxy-2′-fluoro nucleotides:    -   5, 6, 7, 8, 9, 10 or more purine nucleotides in N_(X4) positions        are 2′-O-alkyl nucleotides;    -   5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides in N_(X3)        positions are 2′-deoxy-2′-fluoro nucleotides; and.    -   5, 6, 7, 8, 9. 10 or more purine nucleotides in N_(X3) positions        are 2′-deoxy nucleotides.

In certain embodiments, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule of formula (A); herein

-   -   5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides in N_(X4)        positions are 2′-O-alkyl nucleotides;    -   5, 6, 8, 9, 10 or more purine nucleotides in N_(X4) positions        are ribonucleotides;    -   5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides in N_(X3)        positions are 2′-O-alkyl nucleotides: and    -   5, 6, 7, 8, 9, 10 or more purine nucleotides in N_(X3) positions        are ribonucleotides.

In certain embodiments, the invention features a double-stranded shortinterfering nucleic acid (siNA.) molecule of formula (A); wherein

-   -   5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides in N_(X4)        positions are 2′-deoxy-2′-fluoro nucleotides;    -   5, 6, 7, 8, 9, 10 or more purine nucleotides in N_(X4) positions        are 2′-O-alkyl nucleotides;    -   5, 6, 7, 8, 9, 10 or more pyrimidine. nucleotides: in N_(X3)        positions are 2′-O-alkyl nucleotides; and    -   5, 6, 7, 8, 9, 10 or more purine nucleotides in N_(X3) positions        are 2′-deoxy-2′-fluoro nucleotides.

With respect to any siNA having Formula (A) described herein, in certainembodiments, [N] nucleotides comprise ribonucleotides,deoxyribonucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkylnucleotides, or any combination thereof. In other embodiments, one ormore, e.g., 13, 4, 5, or 6 [N] nucleotide positions optionally comprisea phosphorothioate internucleotide linkage.

With respect to any siNA haying Formula (A) described herein, in certainembodiments, [N] nucleotides are ribonucleotides. In other embodiments,one or more, e.g., 1, 2, 3, 4, 5, or 6 [N] ribonucleotide positionsoptionally comprise a phosphorothioate internucleotide linkage.

With respect to any siNA having Formula (A) described herein, in certainembodiments, [N] nucleotides are 2′-deoxy-2′-fluoro nucleotides. inother embodiments, one or more, e,g., 1, 2, 3, 4, 5, or 6 [N]2′-deoxy-2′-fluoro nucleotide positions optionally comprise aphosphorothioate internucleotide linkage.

With respect to any siNA having Formula (A) described herein, in certainembodiments, [N] nucleotides are 2′-deoxy nucleotides. In otherembodiments, one or more, e.g., 1, 2, 3, 4, 5, or 6 [N] 2′-deoxynucleotide positions optionally comprise a phosphorothioateinternucleotide linkage.

With respect to any siNA having Formula (A) described herein, in certainembodiments, [N] nucleotides are 2′-O-alkyl nucleotides. in otherembodiments, one or more, e.g., 1, 2, 3, 4, 5, or 6 [N] 2′-O-alkylnucleotide positions optionally comprise a phosphorothioateinternucleotide linkage.

With respect to any siNA having Formula (A) described herein, in certainembodiments, X5=3, wherein the three [N] nucleotides of formula (A) arerepresented as 5′-[N1 , N2, N3]-3′, wherein:

-   -   each N1, N2, and N3 is a ribonucleotide; or    -   each N1, N2, and N3 is a 2′-deoxy-2′-fluoro nucleotide; or    -   each N1, N2, and N3 is a 2′-deoxy nucleotide; or    -   each N1, N2, and N3 is a 2′-O-alkyl nucleotide; and    -   any of N1, N2, or N3 optionally comprises a phosphorothioate        internucleotide linkage.

With respect to any siNA having Formula (A) described herein, in certainembodiments, X5=3, wherein the three [N] nucleotides of formula (A) arerepresented as 5′-[N1, N2, N3]-3′, wherein:

-   -   N1 is a 2′-deoxy-2′-fluoro nucleotide, N2 is 2′-deoxy-2′-fluoro        nucleotide, and N3 is a 2′-deoxynucleotide; and    -   any of N1, N2, or N3 optionally comprises a phosphorothioate        internucleotide linkage.

With respect to any siNA having Formula (A) described herein, in certainembodiments, X5=3, wherein the three [N] nucleotides of formula (A) arerepresented as 5′-[N1, N2, N3]-3′, wherein:

-   -   N1 is a 2′-deoxy-2′-fluoro nucleotide, N2 is 2′-deoxy-2′-fluoro        nucleotide, and N3 is a 2′-deoxy-2′-fluoro nucleotide; and    -   any of N1, N2, or N3 optionally comprises a phosphorothioate        internucleotide linkage.

With respect to any siNA having Formula (A) described herein, in certainembodiments, X5=3, wherein the three [N] nucleotides of formula (A) arerepresented as 5′-[N1, N2, .N3]-3′, wherein:

-   -   N1 is a 2′-deoxy nucleotide, N2 is 2′-deoxy-2′-fluoro        nucleotide, and N3 is a 2′-O-alkyl nucleotide; and    -   any of N1, N2, or N3 optionally comprises a phosphorothioate        internucleotide linkage.

With respect to any siNA having Formula (A) described herein, in certainembodiments, X5=3, wherein the three [N] nucleotides of formula (A) arerepresented as 5′-[N1, N2, N3]-3′, wherein:

-   -   N1 is a 2′-deoxy nucleotide, N2 is 2′-deoxy-2′-fluoro        nucleotide, and N3 is a 2′-deoxy nucleotide; and    -   any of N1, N2, or N3 optionally comprises a phosphorothioate        internucleotide linkage.

In certain embodiments of the present invention, double-stranded siNAmolecules are provided that modulate the expression of a target gene viaRNA interference, wherein the molecule has a sense strand and anantisense strand and comprises structure represented by formula (A):

-   -   wherein, the upper strand is the sense strand and the lower        strand is the antisense strand of the double-stranded nucleic        acid molecule; wherein the antisense strand comprises a sequence        having at least 15 nucleotides that are complementary to a        target RNA sequence encoded by the target gene and the sense        strand comprises a sequence that is complementarity to the        antisense strand; each N is independently a nucleotide which is        unmodified or chemically modified or is optionally a        non-nucleotide; each B is independently a terminal cap that is        present or absent; (N) represents overhanging nucleotides:, each        of which is independently unmodified or chemically modified; [N]        represents nucleotides at the 5′-terminus of the antisense        strand; X1 and X2 are independently integers from 0 to 4; X3 is        an integer from 15 to 30; X4 is an integer from 12 to 27; and X5        is an integer from 1-6, provided that the sum of X4 and X5 is an        integer from 15-30; and wherein    -   (a) all pyrimidine nucleotides in N_(X4) positions are        2′-O-alkyl nucleotides;    -   (b) all purine nucleotides in N_(X4) positions are 2′-halo        nucleotides;    -   (c) all pyrimidine nucleotides in N_(X3) positions: are        2′-O-alkyl nucleotides;    -   (d) all purine nucleotides N_(X3) positions are 2′-halo        nucleotides; and    -   (e) [N] position nucleotide(s) are any combination of        ribonucleotides, deoxyribonucleotides, 2′-O-alkyl nucleotides,        or 2′-halo nucleotides;    -   (f) the nucleotide at position 14 from the 5′-end of the        antisense strand is a 2′-deoxy-2′-fluoro nucleotide regardless        of whether it is a purine or pyrimidine; and    -   (g) [N] nucleotides of formula (A) are represented as 5′-[N1,        N2, N3]-3′, wherein        -   i) N1 is a 2′-deoxy nucleotide, N2 is 2′-deoxy-2′-fluoro            nucleotide, and N3 is a 2′-deoxy nucleotide; or        -   ii) N1 is a 2′-deoxy-2′-fluoro nucleotide, N2 is            2′-deoxy-2′-fluoro nucleotide, and N3 is a            2′-deoxy-2′-fluoro nucleotide; or        -   iii) N1 is a 2′-deoxy-2′-fluoro nucleotide, N2 is            2′-deoxy-2′-fluoronucleotide, and N3 is a            2′-deoxynucleotide; or        -   iv) N1 is a 2′-deoxy nucleotide, N2 is 2′-deoxy-2′-fluoro            nucleotide, and N3 is a 2′-O-methyl nucleotide; or        -   v) N1, N2, and N3 are all ribonucleotides having            phosphorothioate internucleotide linkages.

With respect to any siNA having Formula (A) described herein, in certainembodiments, the invention features a double-stranded short interferingnucleic acid (siNA) molecule of formula (A) further comprising one ormore phosphorothioate internucleotide linkages at any N_(X1), N_(X2),N_(X3), N_(X4), or N_(X5) position, or any combination thereof.

In some embodiments, siNA molecules having formula A comprise a terminalphosphate group at the 5′-end of the antisense strand or antisenseregion of the nucleic acid molecule.

In various embodiments, siNA molecules having formula A comprise X5=0,1, 2, or 3; each X1 and X2=1 or 2; X3=18, 19, 20, 21, 22, or 23, and X4=17, 18, 19, 20, 21, 22, or 23.

In certain embodiments, siNA molecules having formula A comprise X5=3.In other embodiments siNA molecules having formula A comprise X5=0.

In certain embodiments, siNA molecules having formula A comprise X1=2and X2=2.

In various embodiments, siNA molecules having formula A comprise X5=0,X1=2, and X2=2. In other embodiments, siNA molecules having formula Acomprise X5=3, X1=2, and X2=2.

In one specific embodiment, an siNA molecule having formula A comprisesX5=3; each X1 and X2=2; X3=19, and X4=16.

In certain embodiments, siNA molecules having formula A comprise caps(B) at the 3′ and 5′ ends of the sense strand or sense region.

In certain embodiments, siNA molecules having formula A comprise caps(B) at the 3′-end of the antisense strand or antisense region.

In various embodiments, siNA molecules having formula A comprise caps(B) at the 3′ and 5′ ends of the sense strand or sense region and caps(B) at the Y.-end of the antisense strand or antisense region.

In yet other embodiments, siNA molecules having formula A comprise caps(B) only at the 5′-end of the sense (upper) strand of thedouble-stranded nucleic acid molecule.

In some embodiments, siNA molecules having formula. A further compriseone or more phosphorothioate internucleotide linkages between thenucleotides. In certain embodiments, siNA molecules having formula Acomprise one or more phosphorothioate internucleotide linkages betweenthe first terminal (N) and the adjacent nucleotide on the 3′end of thesense strand, antisense strand, or both sense strand and antisensestrands of the nucleic acid molecule. For example, a double-strandednucleic acid molecule can comprise X1 and/or X2=2 having overhangingnucleotide positions with a phosphorothioate internucleotide linkage,e.g., (NsN) where “s” indicates phosphorothioate.

In some embodiments, one or more of the nucleotides of siNA moleculeshaving formula A include one or more universal base substitutions.

In some embodiments, one or more of the nucleotides of siNA moleculeshaving formula A include one or more LNA substitutions.

In certain embodiments, siNA molecules having formula A have at position14 from the 5′-end of the antisense strand a ribonucleotide when thenucleotide at that position 14 is a purine.

In certain embodiments, siNA molecules having formula A have at position14 from the 5′-end of the antisense strand a ribonucleotide or a2′-deoxy-2′-fluoro nucleotide when the nucleotide at that position 14 isa purine.

In certain embodiments, siNA molecules having formula A have at position14 from the 5′-end of the antisense strand is a 2′-deoxy-2′-fluoronucleotide when the nucleotide at that position 14 is a pyrimidinenucleotide. In particularly preferred embodiments, position 14 from the5′-end of the antisense strand is a 2′-deoxy-2′-fluoro nucleotideregardless of whether it is a purine or a pyrimidine.

In some embodiments, siNA molecules having formula A comprises (N)nucleotides in the antisense strand (lower strand) that arecomplementary to nucleotides in a target polynucleotide sequence, whichalso has complementarity to the N and [N] nucleotides of the antisense(lower) strand.

Any of the above described modifications, or combinations thereof,discussed above as applicable to siNAs of the invention, including thosein the references cited, can be applied to any of the embodiments tosiNA molecules having formula A.

C. Generation/Synthesis of siNA Molecules

The siNAs of the invention can be obtained using a number of techniquesknown to those of skill in the art. For example the siNA can bechemically synthesized or may be encoded by plasmid (e.g., transcribedas sequences that automatically fold into duplexes with hairpin loops.).siNA can also be generated by cleavage of longer dsRNA (e.g., dsRNAgreater than about 25 nucleotides in length) by the E coli RNase II orDicer. These enzymes process the dsRNA into biologically active siNA(see, e,g., Yang et al., PNAS USA 99:9942-994′7 (2002); Calegari et al.PNAS USA 99:14236 (2002) Byron et al. Ambion Tech Notes; 10 (1):4-6(2009); Kawaski et al., Nucleic Acids Res., 31:981-987 (2003), Knightand Bass, Science, 293:2269-2271 (2001) and Roberston et al., J. Biol.Chem 243:82(1969).

1. Chemical Synthesis

Preferably, siNA of the invention are chemically synthesized.Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al.,1995Nucleic Acids Res, 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end.

siNA molecules without modifications are synthesized using procedures asdescribed in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringeet al., 1990, Nucleic Acids Res., 18, 5433. These syntheses makes use ofcommon nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end thatcan be used for certain siNA molecules of the invention.

In certain embodiments, the siNA molecules of the invention aresynthesized, deprotected, and analyzed according to methods described inU.S. Pat. Nos. 6,995,259, 6,686,463, 6,673,918, 6,649; 751, 6,989,442,and U.S. patent application Ser. No. 10/190,359.

In a non-limiting synthesis example, small scale syntheses are conductedon a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scaleprotocol with a 2.5 min coupling step for 2′-O-methylated nucleotidesand a 45 second coupling step for 2′-deoxy nucleotides or2′-deoxy-2′-fluoro nucleotides. Table 9 outlines the amounts and thecontact times of the reagents used in the synthesis cycle.

Alternatively, the siNA molecules of the present invention can besynthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT Publication No. WO 93/23569; Shabarova et al.,1991Nucleic Acids Research 19, 4247; Beilon et al., 1997, Nucleosides &Nucleotides, 16, 951; Bellon et al., 1., 1997, Bioconjugate Chem. 8,2041, or by hybridization following synthesis and/or deprotection.

Various siNA molecules of the invention can also be synthesized usingthe teachings of Scaringe et al., US Pat. Nos. 5,889,136; 6,008,400; and6,111,086.

D. Carrier/Delivery Systems

The siNA molecules of the invention are added directly, or can becomplexed with cationic lipids, packaged within liposomes, or as arecombinant plasmid or viral vectors which express the siNA molecules,or conjugated with a delivery vehicle, or otherwise delivered to targetcells or tissues. Methods for the delivery of nucleic acid molecules aredescribed in Akhtar et al., 1992, Trends Cell Rio., 2, 139; DeliveryStrategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995,Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang,1999, Handb. Exp. Pharmacal., 137, 165-192; and Lee et al., 2000, ACSSymp. Ser., 752, 184-192. Beigelman et al., U.S. Pat. No. 6,395,713 andSullivan et al., PCT WO 94/02595 further describe the general methodsfor delivery of nucleic acid molecules. These protocols can be utilizedfor the delivery of virtually any nucleic acid molecule. Nucleic acidmolecules can be administered to cells by a variety of methods known tothose of skill in the art, including, but not restricted to,encapsulation in liposomes, by iontophoresis, or by incorporation intoother vehicles, such as biodegradable polymers, hydrogels, cyclodextrins(see for example, Gonzalez et al., 1999, Bioconjugate Chem., 10,1068-1074; Wang et al., International PCT Publication Nos. WO 03/47518and WO 03/46185), poly(lactic-co-glycolic)acid (PWA) and PLCAmicrospheres (see for example U.S. Pat. No. 6,447,796 and US PatentApplication Publication No. US 2002130430), biodegradable nanocapsules,and bioadhesive microspheres, or by proteinaceous vectors (O′Hare andNormand, International PCT Publication No. WO 00/53722).

In one aspect, the present invention provides carrier systems containingthe siNA molecules described herein. In some embodiments, the carriersystem is a lipid-based carrier system, cationic lipid, or liposomenucleic acid complexes, a liposome, a micelle, a virosome, a lipidnanoparticle or a mixture thereof. In other embodiments, the carriersystem is a polymer-based carrier system such as a cationicpolymer-nucleic acid complex. In additional embodiments, the carriersystem is a cyclodextrin-based carrier system such as a cyclodextrinpolymer-nucleic acid complex. In further embodiments, the carrier systemis a protein-based carrier system such as a cationic peptide-nucleicacid complex. Preferably, the carrier system is a lipid nanoparticle(“LNP”) formulation.

In certain embodiments, the siNA molecules of the invention areformulated with a lipid nanoparticle composition such as is described inU.S. patent application Ser. Nos. 11/353,630, 11/586,102, 61/189,295,61/204,878, 61/235,476, 61/249,807, 61/298,022, 61/351,373, 61/317,640,61/345,754, 61/322,054, 12/640,342, and 12/617,079, and PCT ApplicationsNos. PCT/US10/020013 and PCT/US09/053336. In certain preferredembodiments, the siNA molecules of the invention are formulated with alipid nanoparticle composition comprising a cationiclipid/Cholesterol/PEG-C-DMA/DSPC in a 40/48/2/10 ratio or a cationiclipid/Cholesterol/PEG-DMG/DSPC in a 40/48/2/10 ratio. In more certainembodiments, the cationic lipid is DLinDMA, the PEG is PEG-DMG, and theN11.P ratio of the formulation is 2.8. In more preferred embodiments,the cationic lipid is CLinDMA (see U.S. Pat. No. 7,514,099)

In various embodiments, lipid nanoparticle formulations described in anyof the cited applications referred to herein are applied to any siNAmolecule or combination of siNA molecules herein. In some embodiments,the invention features a composition comprising an siNA molecule of theinvention formulated as any of formulation LNP-051; LNP-053; LNP-054;LNP-069; LNP-073; LNP-077; LNP-080; LNP-082; LNP-083; LNP-060; LNP-061;LNP-086; LNP-097; LNP-098; LNP-099; LNP-100; LNP-101; LNP-102; LNP-103;or LNP-104.

In certain other embodiments, the invention features a compositioncomprising an siNA molecule of the invention formulated with any of thecationic lipid formulations described in U.S. Patent Application Nos.61/189,295, 61/204,878, 61/235,476, 61/249,807, 61/298,022, 61/322,054,61/347,640, 61/351,373, 61/382,067, 61/384.486, and 61/388,201.

In other embodiments, the invention features conjugates and/or complexesof siNA molecules of the invention. Such conjugates and/or complexesinclude ligand based and polymer based delivery modalities that can beused to facilitate delivery of siNA molecules into a biological system,such as a cell. The conjugates and complexes provided by the instantinvention can impart therapeutic activity by transferring therapeuticcompounds across cellular membranes, altering the pharmacokinetics,and/or modulating the localization of nucleic acid molecules of theinvention. Non-limiting, examples of such conjugates are described inU.S. Publication Nos. US2008/0152661 1 and US 2004/0162260 A1 (e.g.,CDM-LBA, CDM-Pip-LBA, CDM-PEG, CDM-NAG, etc.) and U.S. patentapplication Ser. Nos. 10/427,160 10/201,394, 61/322,422, 61/378,609, and61/315,223; and U.S. Pat. Nos. 6,528,631; 6,335,434; 6,235,886;6,153,737; 5,214,136; and 5,138,045.

In various embodiments, polyethylene glycol (PEG) can be covalentlyattached to siNA compounds of the present invention. The attached PEGcan be any molecular weight, preferably from about 100 to about 50,000daltons (Da).

In yet other embodiments, the invention features compositions orformulations comprising surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, or long-circulating liposomes orstealth liposomes) and siNA molecules of the invention, such as isdisclosed in for example, International PCT Publication No. WO 96/10391;Ansa et al., International PCT Publication No, WO 96/10390; Holland etal., International PCT Publication No. WO 96/10392.

In sonic embodiments, the siNA molecules of the invention can also beformulated or complexed with polyethyleneiraine and derivatives thereof,such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine(PEI-PEG-GAL) orpolyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives. In one embodiment, the nucleic acidmolecules of the invention are formulated as described in U.S. PatentApplication Publication No. 20030077829.

In other embodiments, siNA molecules of the invention are complexed withmembrane disruptive agents such as those described in U.S, PatentApplication Publication No. 20010007666. In still other embodiments, themembrane disruptive agent or agents and the siNA molecule are alsocomplexed with a cationic lipid or helper lipid molecule, such as thoselipids described in U.S. Pat. No. 6,235,310.

In certain embodiments, siNA molecules of the invention are complexedwith delivery systems as described in U.S. Patent ApplicationPublication Nos. 2003077829; 20050287551; 20050164220; 20050191627;20050118594; 20050153919; 20050085486; and 20030158133; andInternational PCT Publication Nos. WO 00/03683 and WO 02/087541.

In some embodiments, a liposomal formulation of the invention comprisesan siNA molecule of the invention (e.g., siNA) formulated or complexedwith compounds and compositions described in U.S. Pat. Nos. 6,858,224;6,534,484; 6,287,591; 6,835,395; 6,586,410; 6,858,225; 6,815,432;6,586,001; 6,120,798; 6,977,223; 6,998,115; 5,981,501; 5,976,567;5,705,385; and U.S. Patent Application Publication Nos. 2006/0019912;2006/0019258; 2006/0008909; 2005/0255153; 2005/0079212; 2005/0008689;2003/0077829, 2005/0064595, 2005/0175682, 2005/0118253; 2004/0071654;2005/0244504; 2005/0265961 and 2003/0077829.

Alternatively, recombinant plasmids and viral vectors, as discussedabove, which express siNAs of the invention can be used to deliver themolecules of the invention. Delivery of siNA molecule expressing vectorscan be systemic, such as by intravenous or intra-muscularadministration, by administration to target cells ex-planted from asubject followed by reintroduction into the subject, or by any othermeans that would allow for introduction into the desired target cell(for a review see Couture et al., 1996, TIG., 1:2, 510). Suchrecombinant plasmids can also be administered directly or in conjunctionwith a suitable delivery reagents, including, for example, the MintsTransit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin;polycations polylysine) or liposomes lipid-based carrier system,cationic lipid, or liposome nucleic acid complexes, a micelle, avirosome, a lipid nanoparticle.

E. Kits

The present invention also provides nucleic acids in kit form, The kitmay comprise a container. The kit typically contains a nucleic acid ofthe invention with instructions for its administration. In certaininstances, the nucleic acids may have a targeting moiety or deliveryagent attached. Methods of attaching targeting moieties (e.g.antibodies, proteins) or delivery agents (conjugates) are known to thoseof skill in the art. In certain instances, the kit contains more thanone siNA molecule of the invention. The kits may comprise an siNAmolecule of the invention with a pharmaceutically acceptable carrier ordiluent. The kits may further comprise excipients.

F. Therapeutic Uses/Pharmaceutical Compositions

The nucleic acid molecules and pharmaceutical compositions of theinvention can be used to treat diseases, conditions, or phenotypesrelated to gene expression. Non-limiting examples of such diseases,conditions, and phenotypes are described herein and are otherwise knownin the art

1. Indications

Particular conditions and disease states that. can be associated withgene expression modulation include, but are not limited to cancer,proliferative, inflammatory, autoimmune, neurologic, ocular,respiratory, metabolic, dermatological, auditory, liver, kidney,infectious etc. diseases, conditions, or disorders as described hereinor otherwise known in the art, and any other diseases, conditions ordisorders that are related to or will respond to the levels of a target(e.g., target DNA, RNA, protein or polynucleotide) in a cell or tissue,alone or in combination with other therapies.

Proliferative diseases (cancer) include any disease or conditioncharacterized by unregulated cell growth or replication as is known inthe art; including leukemias, for example, acute myelogenous leukemia(AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia(ALL), and chronic lymphocytic leukemia, AIDS related cancers such asKaposi's sarcoma; breast cancers; bone cancers such as Osteosarcoma,Chondrosarcornas, Ewing's sarcoma, Fibrosarcomas, Giant cell tumors,Adamantinomas, and Chordomas; Brain cancers such as Meningiomas,Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, PituitaryTumors, Schwannomas, and Metastatic brain cancers; cancers of the headand neck including various lymphomas such as mantle cell lymphoma,non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngealcarcinoma, gallbladder and bile duct cancers, cancers of the retina suchas retinoblastoma, cancers of the esophagus, gastric cancers, multiplemyeloma, ovarian cancer, uterine cancer, thyroid cancer, testicularcancer, endometrial cancer, melanoma, colorectal cancer, lung cancer,bladder cancer, prostate cancer, lung cancer (including non-small celllung carcinoma), pancreatic cancer, sarcomas, Wilms' tumor, cervicalcancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma,liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladderadeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrugresistant cancers; and proliferative diseases and conditions, such asneovascularization associated with tumor angiogenesis, maculardegeneration (e.g., wet/dry AMD), corneal neovascularization, diabeticretinopathy, neovascular glaucoma, myopic degeneration and otherproliferative diseases and conditions such as restenosis and polycystickidney disease, and any other cancer or proliferative disease,condition, trait, genotype or phenotype that can respond to themodulation of disease related gene expression in a cell or tissue, aloneor in combination with other therapies.

Inflammatory diseases include any disease or condition characterized byan inflammatory or allergic process as is known in the art, such asinflammation, acute inflammation, chronic inflammation, respiratorydisease, atherosclerosis, psoriasis, dermatitis, restenosis, asthma,allergic rhinitis, atopic dermatitis, septic shock, rheumatoidarthritis, inflammatory howl disease, inflammatory pelvic disease, pain,ocular inflammatory disease, celiac disease, Leigh Syndrome, GlycerolKinase Deficiency, Familial eosinophilia (FE), autosomal recessivespastic ataxia, laryngeal inflammatory disease; Tuberculosis, Chroniccholecystitis, Bronchiectasis, Silicosis and other pneumoconioses, andany other inflammatory disease, condition, trait, genotype or phenotypethat can respond to the modulation of disease related gene expression ina cell or tissue, alone or in combination with other therapies.

Autoimmune diseases include any disease or condition characterized byautoimmunity as is known in the art, such as multiple sclerosis,diabetes mellitus, lupus, celiac disease, Crohn's disease, ulcerativecolitis, Guillain-Barre syndrome, sclemderms, Goodpasture's syndrome,Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis,Primary hiliary sclerosis, Sclerosing cholangitis, Autoimmune hepatitis,Addison's disease, Hashimoto's thyroiditis, Fibromyalgia, denier'ssyndrome; transplantation rejection (e.g., prevention of allograftrejection) pernicious anemia, rheumatoid arthritis, systemic lupuserythematosus, dermatomyositis, Sjogrens syndrome, lupus erythematosus,multiple sclerosis, myasthenia gravis, Reiter's syndrome, Grave'sdisease, and any other autoimmune disease, condition, trait, genotype orphenotype that can respond to the modulation of disease related geneexpression in a cell or tissue, alone or in combination with othertherapies.

Infectious diseases include any disease or condition associated with aninfectious agent, such as a virus, bacteria, fungus, prion, or parasite.Non-limiting examples of various viral genes that can be targeted usingsiNA molecules of the invention include Hepatitis C Virus (HCV, forexample GenBank Accession Nos: D11168, D50483.1, L38318 and S82227),Hepatitis B Virus (HBV, for example GenBank Accession No. AF100308.1),Human Immunodeficiency Virus type 1 (HIV-1, for example GenBankAccession No. U51188), Human Immunodeficiency Virus type 2 (HIV-2, forexample GenBank Accession No. X60667), West Nile Virus (WNV for exampleGenBank accession No. NC301.563), cytomegalovirus (CMV for exampleGenBank Accession No. NC_001347), respiratory syncytial virus (RSV forexample GenBank Accession No. NC_001781), influenza virus (for exampleGenBank Accession No. AF03741.2, rhinovirus (for example, GenBankaccession numbers: D00239, X02316, X01087, L24917, M16248, K02121,X01087), papillomavirus (for example GenBank Accession No. NC_001353),Herpes Simplex Virus (HSV for example GenBank Accession No. NC_001345),and other viruses such as HTLV (for example GenBank Accession No.AJ430458). Due to the high sequence variability of many viral genomes,selection of siNA molecules for broad therapeutic applications wouldlikely involve the conserved regions of the viral genome. Nonlimitingexamples of conserved regions of the viral genomes include but are notlimited to 5′-Non Coding Regions (NCR), 3′- Non Coding Regions (NCR)and/or internal ribosome. entry sites (IRES). siNA molecules designedagainst conserved regions of various viral genomes will enable efficientinhibition of viral replication in diverse patient populations and mayensure the effectiveness of the siNA molecules against viral quasispecies which evolve due to mutations in the non-conserved regions ofthe viral genome. Non-limiting examples of bacterial infections includeActinomycosis, Anthrax, Aspergillosis, Bacteremia, Bacterial Infectionsand Mycoses, Bartonella Infections, Botulism, Brucellosis, BurkholderiaInfections, Campylobacter Infections, Candidiasis, Cat-Scratch Disease,Chlamydia Infections, Cholera , Clostridium Infections,Coccidioidomycosis, Cross Infection, Cryptococcosis, Dermatomycoses,Dermatomycoses, Diphtheria, Ehrlichiosis, Escherichia coli Infections,Fasciitis, Necrotizing, Fusobacterium infections, Gas Gangrene,Grain-Negative Bacterial Infections, Gram-Positive Bacterial infections,Histoplasmosis, Impetigo, Klebsiella Infections, Legionellosis, Leprosy,Leptospirosis, Listeria Infections, Lyme Disease, Maduromycosis,Melioidosis, Mycobacterium Infections, Mycoplasma Infections, Mycoses,Nocardia Infections, Onychomycosis, Ornithosis, Plague, PneurnococcalInfections, Pseudomonas Infections, Q Fever, Rat-Bite Fever, RelapsingFever, Rheumatic Fever, Rickettsia Infections, Rocky Mountain SpottedFever, Salmonella infections, Scarlet Fever, Scrub Typhus, Sepsis,Sexually Transmitted Diseases - Bacterial, Bacterial Skin Diseases,Staphylococcal Infections, Streptococcal Infections, Tetanus, Tick-BorneDiseases, Tuberculosis, Tularemia, Typhoid Fever, Typhus, EpidemicLouse-Borne, Vibrio Infections, Yaws, Yersinia Infections, Zoonoses, andZygomycosis. Non-limiting examples of fungal infections includeAspergillosis, Blastomycosis, Coccidioidomycosis, Cryptococcosis, FungalInfections of Fingernails and Toenails, Fungal Sinusitis,Histoplasmosis, Histoplasmosis, Mucormycosis, Nail Fungal Infection,Paracoccidioidomycosis, Sporotrichosis, Valley Fever(Coccidioidomycosis), and Mold Allergy.

Neurologic diseases include any disease or condition affecting thecentral or peripheral nervous system, including ADHD, AIDS -Neurological Complications, Absence of the Septum Pellucidum, AcquiredEpileptiform Aphasia, Acute Disseminated Encephalomyelitis,Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, AicardiSyndrome, Alexander Disease, Alpers' Disease, Alternating Hemiplegia,Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Anencephaly,Aneurysm, Angelman Syndrome, Angiomatosis, Anoxia, Aphasia, Apraxia,Arachnoid Cysts, Arachnoiditis, Amold-Chiari Malformation, ArteriovenousMalformation, Aspartame, Asperger Syndrome, Ataxia Telangiectasia,Ataxia, Attention Deficit-Hyperactivity Disorder, Autism, AutonomicDysfunction, Back Pain, Barth Syndrome, Batten Disease, Behcet'sDisease, Bell's Palsy, Benign Essential Blepharospasm, Benign FocalAmyotrophy, Benign intracranial Hypertension, Bernhardt-Roth Syndrome,Binswanger's Disease, Blepharospasm, Bloch-Sulzberger Syndrome, BrachialPlexus Birth Injuries:, Brachial Plexus Injuries, Bradbury-EgglestonSyndrome, Brain Aneurysm, Brain Injury, Brain and Spinal Tumors,Brown-Sequard Syndrome, Bulbospinal Muscular Atrophy, Canavan Disease,Carpal Tunnel Syndrome, Causalgia, Cavernomas, Cavernous Angioma,Cavernous Malformation, Central Cervical Cord Syndrome, Central CordSyndrome, Central Pain Syndrome, Cephalic Disorders, CerebellarDegeneration, Cerebellar Hypoplasia, Cerebral Aneurysm, CerebralArteriosclerosis, Cerebral Atrophy, Cerebral Beriberi, CerebralGigantism, Cerebral Hypoxia, Cerebral Palsy,Cerebro-Oculo-Facio-Skeletal Syndrome, Charcot-Marie-Tooth Disorder,Chiari Malformation, Chorea, Choreoacanthocytosis, Chronic InflammatoryDemyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance,Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, Coma,including Persistent Vegetative State, Complex Regional Pain Syndrome,Congenital Facial Diplegia, Congenital Myasthenia, Congenital Myopathy,Congenital Vascular Cavernous Malformations, Cordcobasal Degeneration,Cranial Arteritis, Craniosynostosis, Creutzfeledt-JakOb Disease,Cumulative Trauma Disorders, Cushing's Syndrome, Cytomegalic InclusionBody Disease (CIBD), Cytomegalovirus Infection, Dancing Eyes-DancingFeet Syndrome, Dandy-Walker Syndrome, Dawson Disease, De Morsier'sSyndrome, Dejerine-Klumpke Palsy, Dementia—Muld-Infarct,Dementia—Subcortical, Dementia With Lewy Bodies, Dermatomyositis,Developmental Dyspraxia, Devic's Syndrome, Diabetic Neuropathy, DiffuseSclerosis, Dravet's Syndrome, Dysautonomia, Dysgraphia, Dyslexia,Dysphagia, Dyspraxia, Dystonias, Early infantile EpilepticEncephalopathy, Empty Sella Syndrome, Encephalitis Lediargica,Encephalitis and Meningitis, Encephaloceles, Encephalopathy,Encephalotrigeminal Angiomatosis, Epilepsy, Erb's Palsy, Erb-Duchenneand Dejerine-Klutnpke Palsies, Fabry's Disease, Fakir's: Syndrome,Fainting, Familial Dysautonomia, Familial Hemangioma, Idiopathic BasalGanglia Calcification, Familial Spastic Paralysis, Febrile Seizures(e.g., GEFS and. GEFS plus), Fisher Syndrome, Floppy Infant Syndrome,Friedreich's Ataxia, Gaucher's Disease, Gerstmann's Syndrome,Gerstmann-Straussler-Scheinker Disease, Giant Cell Arteritis, Giant CellInclusion Disease, Globoid Cell Leukodystrophy, GlossopharyngealNeuralgia, Guillain-Barre Syndrome, HTLV-1 Associated Myelopathy,Hallervorden-Spatz Disease, Head Injury, Headache, Hemicrania Continua,Hemifacial Spasm, Hemiplegia Aiterans, Hereditary Neuropathies,Hereditary Spastic Paraplegia, Heredopathia Atactica Polyneuritiformis,Herpes Zoster Oticus, Herpes Zoster, Hirayama Syndrome,Holoprosencephaly, Huntington's Disease, Hydranencephaly,Hydrocephalus—Normal Pressure, Hydrocephalus, Hydromyelia,Hypercortisolism, Hypersorania, Hypertonia, Hypotonia, Hypoxia,immune-Mediated Encephalomyelitis, Inclusion Body Myositis,Incontinentia Pigmenti., Infantile Hypotonia, Infantile Phytanic AcidStorage Disease, Infantile Refsum Disease, Infantile Spasms,inflammatory Myopathy, Intestinal Lipodystrophy, Intracranial Cysts,intracranial Hypertension, Isaac's Syndrome, Joubert Syndrome,Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne syndrome,Kleine-Levin syndrome, Klippel Feil Syndrome, Klippel-Trenaunay Syndrome(KTS), Kltiver-Bucy Syndrome, Korsakoff s Aninesic Syndrome, KrabbeDisease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton MyasthenicSyndrome, Landau-Kleffner Syndrome, LateTal Femoral Cutaneous NerveEntrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh'sDisease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy,Levine-Critchley Syndrome, Lewy Body Dementia, Lissencephaly, Locked-InSyndrome, Lou Gehrig's Disease, Lupus—Neurological Sequelae, LymeDisease—Neurological Complications, Machado-Joseph Disease,Macrencephaly, Megalencephaly, Melkersson-Rosenthal Syndrome,Meningitis, Menkes Disease, Meralgia Paresthetica, MetachromaticLeukodystrophy, Microcephaly, Migraine, Miller Fisher Syndrome,Mini-Strokes, Mitochondrial Myopathies, Mobius Syndrome, MonornelicAmyotrophy, Motor Neuron Diseases, Moyamoya Disease, Mucolipidoses,Mucopolysaccharidoses, Multi-Infarct Dementia, Multifocal MotorNeuropathy, Multiple Sclerosis, Multiple System Atrophy with OrthostaticHypotension, Multiple System Atrophy, Muscular Dystrophy,Myasthenia—Congenital, Myasthenia Gravis, Myelinoclastic DiffuseSclerosis, Myoclonic Encephalopathy of Infants, Myoclonus,Myopathy—Congenital, Myopathy Thyrotoxic, Myopathy, Myotonia Congenita,Myotonia, Narcolepsy, Neuroacanthocytosis, Neurodegeneration with BrainIron Accumulation, Neurofibromatosis, Neuroleptic Malignant Syndrome,Neurological Complications of AIDS, Neurological Manifestations of PompeDisease, Neuromyelitis Optica, Neuromyotonia, Neuronal CeroidLipofuscinosis, Neuronal Migration Disorders, Neuropathy—Hereditary,Neurosarcoidosis, Neurotoxicity, Nevus Cavernosus, Niemann-Pick Disease,O'Sullivan-McLeod Syndrome, Occipital Neuralgia, Occult SpinalDysraphism Sequence, Ohtahara Syndrome, Olivopontocerebellar Atrophy,Opsoclonus Myoclonus, Orthostatic Hypotension, Overuse Syndrome,Pain—Chronic, Paraneoplastic Syndromes, Paresthesia, Parkinson'sDisease, Parmyotonia Congenita, Paroxysmal Choreoathetosis, ParoxysmalHemicrania, Parry-Romberg, Pelizaeus-Merzbacher Disease, Pena Shokeir IISyndrome, Perineural Cysts, Periodic Paralyses, Peripheral Neuropathy,Periventricular Leukomalacia, Persistent Vegetative State, PervasiveDevelopmental Disorders, Phytanic Acid Storage Disease, Pick's Disease,Pirifortnis Syndrome, Pituitary Tumors, Polymyositis, Pompe Disease,Porencephaly, Post-Polio Syndrome, Postherpetic Neuralgia,Postinfectious Encephalomyelitis, Postural Hypotension, PosturalOrthostatic Tachycardia Syndrome, Postural Tachycardia Syndrome, PrimaryLateral Sclerosis, Prion Diseases, Progressive Hemifacial Atrophy,Progressive Locomotor Ataxia, Progressive MultifocalLeukoencephalopathy, Progressive Sclerosing Poliodystrophy, ProgressiveSupranuclear Palsy, Pseudotumor Cerebri, Pyridoxine Dependent andPyridoxine Responsive Siezure Disorders, Ramsay Hunt Syndrome Type I,Ramsay Hunt Syndrome Type II, Rasmussen's Encephalitis and otherautoimmune epilepsies, Reflex Sympathetic Dystrophy Syndrome, RefsumDisease - Infantile, Refsum Disease, Repetitive Motion Disorders,Repetitive Stress Injuries, Restless Legs Syndrome,Retrovirus-Associated Myelopathy, Rett Syndrome, Reye's Syndrome,Riley-Day Syndrome, SUNCT Headache, Sacral Nerve Root Cysts, Saint VitusDance, Salivary Gland Disease, Sandhoff Disease, Schilder's Disease,Schizencephaly, Seizure Disorders, Septo-Optic Dysplasia, SevereMyoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome, Shingles,Shy-Drager Syndrome, Sjogren's Syndrome, Sleep Apnea, Sleeping Sickness,Soto's Syndrome, Spasticity, Spina Bifida, Spinal Cord Infarction,Spinal Cord Injury, Spinal Cord Tumors, Spinal Muscular Atrophy,Spinocerebellar Atrophy, Steele-Richardson-Olszewski Syndrome,Stiff-Person Syndrome, Striatonigral Degeneration, Stroke, Sturge-WeberSyndrome, Subacute Sclerosing Panencephalitis, Subcortical.Arteriosclerotic Encephalopathy, Swallowing Disorders, Sydenham Chorea,Syncope, Syphilitic Spinal Sclerosis, Syringohydromyelia, Syringomyelia,Systemic Lupus Erythematosus, Tabes Dorsalis, Tardive Dyskinesia, TarlovCysts, Tay-Sachs Disease, Temporal Arteritis, Tethered Spinal CordSyndrome, Thomsen Disease, Thoracic Outlet. Syndrome, ThyrotoxicMyopathy, Tic Douloureux, Todd's Paralysis, Tourette Syndrome,Transient. Ischemic Attack, Transmissible Spongiform Encephalopathies,Transverse Myelitis, Traumatic Brain Injury, Tremor, TrigeminalNeuralgia, Tropical Spastic Paraparesis, Tuberous Sclerosis, VascularErectile Tumor, Vasculitis including Temporal Arteritis, Von Economo'sDisease, Von Hippel-Lindau disease (VHL), Von Recklinghausen's Disease,Wallenberg's Syndrome, Werdnig-Hoffman Disease, Wernicke-KorsakoffSyndrome, West Syndrome, Whipple's Disease, Williams Syndrome, Wilson'sDisease, X-Linked Spinal and Bulbar Muscular Atrophy, and ZellwegerSyndrome.

Respiratory diseases include any disease or condition affecting therespiratory tract, such as asthma, chronic obstructive pulmonary diseaseor “COPD”, allergic rhinitis, sinusitis, pulmonary vasoconstriction,inflammation, allergies, impeded respiration, respiratory distresssyndrome, cystic fibrosis, pulmonary hypertension, pulmonaryvasoconstriction, emphysema, and any other respiratory disease,condition, trait, genotype or phenotype that can respond to themodulation of disease related gene expression in a cell or tissue, aloneor in combination with other therapies.

Ocular diseases include any disease or condition affecting eye andrelated structures as is known in the art, such as Cystoid MacularEdema, Asteroid Hyalosis, Pathological Myopia and Posterior Staphyloma,Toxocariasis (Ocular Larva Migrans), Retinal Vein Occlusion, PosteriorVitreous Detachment, Tractional Retinal Tears, Epiretinal Membrane,Diabetic Retinopathy, Lattice Degeneration, Retinal Vein Occlusion,Retinal Artery Occlusion, Macular Degeneration (e.g., age relatedmacular degeneration such as wet AMD or dry AMD), Toxoplasmosis,Choroidal Melanoma, Acquired Retinoschisis, Hollenhorst Plaque,Idiopathic Central Serous Chorioretinopathy, Macular Hole, PresumedOcular Histoplasmosis Syndrome, Retinal Macroaneursy RetinitisPigmentosa, Retinal Detachment, Hypertensive Retinopathy, RetinalPigment Epithelium (RPE) Detachment, Papillophlebitis, Ocular IschemicSyndrome, Coats' Disease, Leber's Miliary Aneurysm, ConjunctivalNeoplasms, Allergic Conjunctivitis, Vernal Conjunctivitis, AcuteBacterial Conjunctivitis, Allergic Conjunctivitis &VernalKeratoconjunctivitis, Viral Conjunctivitis, Bacterial Conjunctivitis,Chlamydial & Gonococcal Conjunctivitis, Conjunctival Laceration,Episcleritis, Scleritis, Pingueculitis, Pterygium, Superior LimbicKeratoconjunctivitis (SLK of Theodore), Toxic Conjunctivitis,Conjunctivitis with Pseudomembrane, Giant Papillary Conjunctivitis,Terrien's Marginal Degeneration, Acanthamoeba Keratitis, FungalKeratitis, Filamentary Keratitis, Bacterial Keratitis, KeratitisSicca/Dry Eye Syndrome, Bacterial Keratitis, Herpes Simplex Keratitis,Sterile Corneal Infiltrates, Phlyctenulosis, Corneal Abrasion &Recurrent Corneal Erosion, Corneal Foreign Body, Chemical Burs,Epithelial Basement Membrane Dystrophy (EBMD), Thygeson's SuperficialPunctate Keratopathy, Corneal Laceration, Salzmann's NodularDegeneration, Fuchs' Endothelial Dystrophy, Crystalline LensSubluxation, Ciliary-Block Glaucoma, Primary Open-Angle Glaucoma,Pigment Dispersion Syndrome and Pigmentary Glaucoma, PseudoexfoliationSyndrom and Pseudoexfoliative Glaucoma, Anterior Uveitis, Primary OpenAngie Glaucoma, uveitic Glaucoma & Glaucomatocyclitic Crisis, PigmentDispersion Syndrome & Pigmentary Glaucoma, Acute Angle Closure Glaucoma,Anterior Uveitis, Hyphema, Angle Recession Glaucoma, Lens InducedGlaucoma, Pseudoexfoliation Syndrome and Pseudoexfoliative Glaucoma,Axenfeld-Rieger Syndrome, Neovascular Glaucoma, Pars Planitis, ChoroidalRupture, Duane's Retraction Syndrome, Toxic/Nutritional OpticNeuropathy, Aberrant Regeneration of Cranial Nerve III, IntracranialMass Lesions, Carotid-Cavernous Sinus Fistula, Anterior Ischemic OpticNeuropathy, Optic Disc Edema & Papillederaa, Cranial Nerve III Palsy,Cranial Nerve TV Palsy, Cranial Nerve VI Palsy, Cranial Nerve VII(Facial Nerve) Palsy, Homer's Syndrome, Internuclear Ophthalmoplegia,Optic Nerve Head Hypoplasia, Optic Pit, Tonic Pupil, Optic Nerve HeadDrusen, Demyelinating Optic Neuropathy (Optic Neuritis, RetrobulbarOptic Neuritis), Amaurosis Fugax and Transient Ischemic Attack,Pseudotumor Cerebri, Pituitary Adenoma, Molluscum Contagiosum,Canaliculitis, Verruca and Papilloma, Pediculosis and Pthiriasis,Blepharitis, Hordeolum, Preseptal Cellulitis, Chalazion, Basal CellCarcinoma, Herpes Zoster Ophthalmicus, Pediculosis & Phthiriasis,Blow-out Fracture, Chronic Epiphora, Dacryocystitis, Herpes SimplexBlepharitis, Orbital Cellulitis, Senile Entropion, and Squamous CellCarcinoma.

Dermatologic diseases include any disease or condition affecting theskin, dermis, or any substructure therein such as hair, follicle, etc.Dermatological diseases, disorders, conditions, and traits can includepsoriasis, ectopic dermatitis, skin cancers such as melanoma and basalcell carcinoma, hair loss, hair removal, alterations in pigmentation,and any other disease, condition, or trait associated with the skin,dermis, or structures therein.

Auditory diseases include any disease or condition affecting theauditory system, including the ear, such as the inner ear, middle ear,outer ear, auditory nerve, and any substructures therein. Auditorydiseases, disorders, conditions, and traits can include hearing loss,deafness, tinnitus, Meniere's Disease, vertigo, balance and motiondisorders, and any other disease, condition, or trait associated withthe ear, or structures therein.

Metabolic diseases include any disease or condition affecting metabolicpathways as in known in the art. Metabolic disease can result in anabnormal metabolic process, either congenital due to inherited enzymeabnormality (inborn errors of metabolism) or acquired due to disease ofan endocrine organ or failure of a metabolically important organ such asthe liver. In one embodiment, metabolic disease includes hyperlipidemia,hypercholesterolemia, cardiovascular disease, atherosclerosis,hypertension, diabetes (e.g., type I and/or type II diabetes), insulinresistance, and/or obesity.

Cardiovascular diseases include any disease or condition affecting theheart and vasculature, including but not limited to, coronary heartdisease (CHD), cerebrovascular disease (CVD), aortic stenosis,peripheral vascular disease, atherosclerosis, arteriosclerosis,myocardial infarction (heart attack), cerebrovascular diseases (stroke),transient ischemic attacks (TIA), angina (stable and unstable), atrialfibrillation, arrhythmia, vavular disease, congestive heart failure,hypercholoesterolemia, type I hyperlipoproteinemia, type IIhyperlipoproteinemia, type III hyperlipoproteineraia, type IVhyperlipoproteinemia, type V hyperlipoproteinemia, secondaryhypertrigliceridemia, and familial lecithin cholesterol acyltransferasedeficiency.

It, is understood that the siNA molecules of the invention can silencethe. expression of target genes and thus amenable to the treatment ofvarious diseases and conditions herein or otherwise known in the art.Treatment of a disease can be evaluated by directly measuring theprogress of the disease in a subject. It can also be inferred throughobserving a change or reversal in a condition associated with thedisease. Additionally, the siNA molecules of the invention can be usedas a prophylaxis. Thus, the use of the nucleic acid molecules andpharmaceutical compositions of the invention can be used to ameliorate,treat, prevent, and/or cure these diseases and others associated withgene expression and/or activity.

Subjects (e.g., mammalian, human) that are amendable for treatment usingthe siNA molecules of the invention (optionally further substituted ormodified or conjugated), compositions thereof, and methods of thepresent disclosure include those suffering from one or more disease orcondition mediated, at least in part, by an aberrant expression level ofthe target gene or sequence, those at risk of developing a diseasecaused by or associated with the aberrant levels of a targetgene/sequence, or those which are amenable to treatment by replenishingor increasing the level of RNAi. mediated by the corresponding siNAmolecule, including a hyperproliferative (e.g., cancer), angiogenic,metabolic, or inflammatory (e.g., arthritis) disease or disorder orcondition.

Compositions and methods disclosed herein are useful in the treatment ofa wide variety of target viruses, including retrovirus, such as humanimmunodeficiency virus (HIV), Hepatitis C Virus, Hepatitis B Virus,Coronavirus, as well as respiratory viruses, including human RespiratorySyncytial Virus, human IVIetapneumovirus, human Parainfluenza virus,Rhinovirus and Influenza virus.

In other examples, the compositions and methods of this disclosure areuseful as therapeutic tools to treat or prevent symptoms of, forexample, hyperproliferative disorders. Exemplary hyperproliferativedisorders include neoplasms, carcinomas, sarcomas, tumors, or cancer.More exemplary hyperproliferative disorders include oral cancer, throatcancer, laryngeal cancer, esophageal cancer, pharyngeal cancer,nasopharyngeal cancer, oropharyngeal cancer, gastrointestinal tractcancer, gastrointestinal stromal tumors (GIST), small intestine cancer,colon cancer, rectal cancer, colorectal cancer, anal cancer, pancreaticcancer, breast cancer, cervical cancer, uterine cancer, vulvar cancer,vaginal cancer, urinary tract cancer, bladder cancer, kidney cancer,adrenocortical cancer, islet cell carcinoma, gallbladder cancer, stomachcancer, prostate cancer, ovarian cancer, endometrial cancer,trophoblastic tumor, testicular cancer, penial cancer, bone cancer,osteosarcoma, liver cancer, extrahepatic bile duct cancer, skin cancer,basal cell carcinoma (BCC), lung cancer, small cell lung cancer,non-small cell lung cancer (NSCLC), brain cancer, melanoma, Kaposi'ssarcoma, eye cancer, head and neck cancer, squamous cell carcinoma ofhead and neck, tymoma, thymic carcinoma, thyroid cancer, parathyroidcancer, Hippel-Lindau syndrome, leukemia, acute myeloid leukemia,chronic myelogenous leukemia, acute lymphoblastic leukemia, hairy cellleukemia, lymphoma, non- Hodgkin's lymphoma, Burkitt's lymphoma, T-celllymphoma, multiple myeloma, malignant pleural mesothelioma, Barrett'sadenocarcinoma, Wilm's tumor, or the like. In other examples, thecompositions and methods of this disclosure are useful as therapeutictools to regulate expression of one or more target gene to treat orprevent symptoms of, for example, inflammatory disorders, Exemplaryinflammatory disorders include diabetes mellitus, rheumatoid arthritis,pannus growth in inflamed synovial lining, collagen-induced arthritis,spondylarthritis, ankylosing spondylitis, multiple sclerosis,encephalomyelitis, inflammatory bowel disease, Crohn's disease,psoriasis or psoriatic arthritis, myasthenia gravis, systemic lupuserythematosis, graft-versus-host disease, atherosclerosis, andallergies.

Other exemplary disorders that can be treated with the siNA molecules,compositions and methods of the instant disclosure include metabolicdisorders, cardiac disease, pulmonary disease, neovascularization,ischemic disorders, age-related macular degeneration, diabeticretinopathy, glomerulonephritis, diabetes, asthma, chronic obstructivepulmonary disease, chronic bronchitis, lymphangiogenesis, andatherosclerosis.

2. Pharmaceutical Compositions

The siNA molecules of the instant invention provide useful reagents andmethods for a variety of therapeutic, prophylactic, cosmetic,veterinary, diagnostic, target validation, genomic discovery, geneticengineering, and pharmacogenornic applications,

a. Formulations

Thus, the present invention, in one aspect, also provides forpharmaceutical compositions of the siNA molecules of the invention,i.e., compositions in a pharmaceutically acceptable carrier or diluent.These pharmaceutical compositions include salts, esters, or salts ofsuch esters, of the above compounds, e.g., acid addition salts, forexample, salts of hydrochloric, hydrobromic, hydroiodic, acetic acid,and benzene sulfonic acid. Other salts include for example, sodium,potassium, manganese, ammonium, and calcium salts. These formulations orcompositions can comprise a pharmaceutically acceptable carrier ordiluent as is generally known in the art. The pharmaceuticalcompositions of the present disclosure are formulated to all the siNAmolecule(s) described herein to be bioavailable upon administration to asubject.

In one embodiment, the invention features a pharmaceutical compositioncomprising any siNA comprising formula (A) as described herein.

The siNA molecules of the invention are preferably formulated aspharmaceutical compositions prior to administering to a subject,according to techniques known in the art. Pharmaceutical compositions ofthe present invention are characterized as being at least sterile andpyrogen-free. Methods for preparing pharmaceutical compositions of theinvention are within the skill in the art for example as described inRemington's Pharmaceutical Science, 21^(st) ed., Mack PublishingCompany, Easton, Pa., A. R. Gennaro edit., 2005.

In some embodiments, pharmaceutical compositions of the invention (e.g.siNA(s) and/or LNP formulations or conjugates or other deliveryformulations thereof) further comprise conventional pharmaceuticalexcipients and/or additives. Suitable pharmaceutical excipients includepreservatives, flavoring agents, stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude physiologically biocompatible buffers (e.g., trimethylaminehydrochloride), addition of chelants (such as, for example, DTPA orDTPA-bisaraide) or calcium chelate complexes (as for example calciumDTPA, C7aNaDTPA-bisamide), or, optionally, additions of calcium orsodium salts (for example, calcium chloride, calcium ascorbate, calciumgluconate or calcium lactate). In addition, antioxidants and suspendingagents can be used.

Non-limiting examples of various types of formulations for localadministration include ointments, lotions, creams, gels, foams,preparations for delivery by transderinal patches, powders, sprays,aerosols, capsules or cartridges for use in an inhaler or insufflator ordrops (for example eye or nose drops), solutions/suspensions fornebulization, suppositories, pessaries, retention enemas and chewable orsuckabie tablets or pellets (for example for the treatment of aphthousulcers) or liposome or inicroencapsulation preparations.

Ointments, creams and gels, can, for example, can be formulated with anaqueous or oily base with the addition of suitable thickening and/orgelling agent and/or solvents. Non limiting examples of such bases canthus, for example, include water and/or an oil such as liquid paraffinor a vegetable oil such as arachis oil or castor oil, or a solvent suchas polyethylene glycol. Various thickening agents and gelling agents canbe used depending on the nature of the base. Non-limiting examples ofsuch agents include soft paraffin, aluminum stearate, cetostearylalcohol, polyethylene glycols, woolfat, beeswax, carboxypolymethyleneand cellulose derivatives, and/or glyceryl monostearate and/or non-ionicemulsifying agents.

In one embodiment lotions can be formulated with an aqueous or oily baseand will in general also contain one or more emulsifying agents,stabilizing agents, dispersing agents, suspending agents or thickeningagents.

In one embodiment powders for external application can be formed withthe aid of any suitable powder base, for example, talc, lactose orstarch. Drops can be formulated with an aqueous or non-aqueous base alsocomprising one or more dispersing agents, solubilizing agents,suspending agents or preservatives.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropromj-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate; or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample. heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin,

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono-or diglycerides. in addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The nucleic acid molecules of the invention can also be administered inthe form of suppositories, e.g., for rectal administration of the drug.These compositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterallyin a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

In other embodiments, the siNA and LNP compositions, or conjugates, andor delivery formulations provided herein for use in pulmonary deliveryfurther comprise one or more surfactants. Suitable surfactants orsurfactant components f6r enhancing the uptake of the compositions ofthe invention include synthetic and natural as well as full andtruncated forms of surfactant protein A, surfactant protein B,surfactant protein C, surfactant protein D and surfactant Protein E,di-saturated phosphatidylcholine (other than dipalmitoyl),dipalmitoylphosphatidylcholine, phosphatidylcholine,phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine,phosphatidylserine; phosphatidic acid, ubiquinones,Iysophosphatidylethanolamine, lysophosphatidylcholine,palmitoyl-lysophosphatidylcholine, dehydroepiandrosterone, dolichols,sulfatidic acid, glycerol-3-phosphate, dihydroxyacetone phosphate,glycerol, glycero-3-phosphocholine, dihydroxyacetone, palmitate,cytidine diphosphate (CDP) diacylglycerol, CDP choline, choline, cholinephosphate; as well as natural and artificial lamellar bodies which arethe natural carrier vehicles for the components of surfactant, omega-3fatty acids, polyenic acid, polyenoic acid, lecithin, palmitinic acid,non-ionic block copolymers of ethylene or propylene oxides,polyoxypropylene, monomeric and polymeric, polyoxyethylene, monomericand polymeric, poly (vinyl amine) with dextran and/or alkanoyl sidechains, Brij 35, Triton X-100 and synthetic surfactants ALEC, Exosurf,Survan and Atovaquone, among others. These surfactants can be usedeither as single or part of a multiple component surfactant in aformulation, or as covale,ntly bound additions to the 5′ and/or 3′ endsof the nucleic acid component of a pharmaceutical composition herein.

b. Combinations

The siNAs and pharmaceutical formulations according to the invention canbe administered to a subject alone or used in combination with orinclude one or more other therapeutic agents, for example, antiviral oranticancer agents. Thus, combinations of the presently disclosedcompounds with other antiviral or anti-cancer or chemotherapeutic agentsare within the scope of the invention

Examples of anti-cancer or chemotherapeutic agents can be found inCancer Principles and Practice of Oncology by V. I. Devita and S.Hellman (editors), 6^(th) edition (February 15, 2001), LippincottWilliams & Wilkins Publishers. A person of ordinary skill in the artwould be able to discern which combinations of agents would be usefulbased on the particular characteristics of the drugs and the cancerinvolved. Such anti-cancer agents include, but are not limited to, thefollowing: estrogen receptor modulators, androgen receptor modulators,retinoid receptor modulators, cytotoxic/cytostatic agents,antiproliferative: agents, prenyl-protein transferase inhibitors,HMG-CoA reductase inhibitors and other angiogenesis inhibitors,inhibitors of cell proliferation and survival signaling, apoptosisinducing agents and agents that interfere with cell cycle checkpoints.

In a further embodiment, therefore, the invention provides a combinationcomprising an siNA molecule of the invention or a pharmaceuticallyacceptable salt, solvate or physiologically functional derivativethereof together with one or more therapeutic agents as described hereinor as is otherwise known in the art.

Examples of estrogen receptor modulators that can be used in combinationwith the compounds of the invention include, but are not limited to,tamoxifen, raloxifene, idoxifene, LY353381, LY117081, toremifene,fulvestrant, 4[7-(2,2-dimethyl- 1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3- yl]-phenyl-2,2-dimethylpropanoate,4,4′-dihydroxybenzophenone-2,4-dinitrophenvl-hydrazone, and SH646.

Examples of androgen receptor modulators that can be used in combinationwith the compounds of the invention include, but are not limited to,finasteride and other 5α-reductase inhibitors, nilutamide, flutamide,hicalutamide, liarozole, and abiraterone acetate,

Examples of such retinoid receptor modulators that can be used incombination with the compounds of the invention include, but are notlimited to, bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoicacid, α-difluoromethylomithine, ILX23-7553, trans-N-(4′-hydroxyphenyl)retinainide, and N-4-carboxyphenyl retinamide.

Examples of cytotoxic agents that can be used in combination with thecompounds of the invention include, but are not limited to, sertenef,cachectin, ifosfamide, tasonermin, ionidamine, carboplatin, altretamine,prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin,oxaliplatin, temozolomide heptaplatin, estramustine, improsulfantosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa,lohaplatin, satraplatin, profiromycin, cisplatin, irofulven,dexifosfaraide, cis-aminedichloro(2-methyl-pyridine)platinum,benzylguanine, glufosfamide, GPX100, (trans, trans,trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum(II)]tetrachloride,diarizidinylspermine, arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin,pinafide, valrubicin, amrubicin, antineoplaston,3′-deamino-3′-morpholino- 13-deoxo-10-hydroxycarminomycin, annamycin,galarubicin, elinafide, MEN10755, and4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin (seeWO 00/50032).

An example of a hypoxia activatable compound that can be used incombination with the compounds of the invention is tirapazamine.

Examples of proteasome inhibitors that can be used in combination withthe compounds of the invention include, but are not limited to,lactacystin and bortezomib.

Examples of microtubule inhibitors/microtubule-stabilising agents thatcan be used in combination with the compounds of the invention include,but are not limited to, paclitaxel, vindesine sulfate,3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine, docetaxol, rhizoxin,dolastat.in, mivobulin isethionate, auristatin, cemadotin, RPRI09881,BMS184476, vinflunine, cryptophycin,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl) benzene sulfonamide,anhydrovinblastine, N,Ndimethyl-L-valyl-L-valyl-L-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide,TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and6,288,237) and BMS188797.

Some examples of topoisomerase inhibitors that can be used incombination with the compounds of the invention include, but are notlimited to, are topotecan, hycaptamine, irinotecan, rubitecan,6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin,9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H)propanamine, 1-amino-9-ethyl-5-fluoro-2,3″-dihydro-9-hydroxy -4-methyl-1H, 12H-benzo[de]pyrano[3′,4′:b,7]-indolizino[1,2b]quinoline-10,13(9H,15H)dione, lurtotecan,7-[2-(N-isopropylamino)ethyl]-(20S)caraptothecin, BNP1350, BNPI1100,BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane,2′-dimethylamino-2′-deoxy-etoposide, GL331,N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide,asulacrine, (5a, 5aB,8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydroOxy-3,5-dimalioxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3′,4′:6,7)naphtho(2,3-d)-1,3-dioxo1-6-one,2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium,6,9-bis[(2-aminoethyl)amitio]benzo[g]isoguinoline-5,10-dione,5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one,N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide, N-(2-(dimethylamino)ethyl)acridine-4-carboxamide,6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one,and dimesna.

Examples of inhibitors of mitotic kinesins, and in particular the humanmitotic kinesin KSP, that can be used in combination with the compoundsof the invention include, but are not limited to, inhibitors describedin PCT Publications WO 01/30768, WO 01/98278, WO 03/050,064, WO03/050,122, WO 03/049,527, WO 03/049,679, WO 03/049,678, WO04/039774,WO03/079973, WO03/099211, WO03/105855, WO03/106417, WO04/037171,WO04/058148, WO04/058700, WO04/126699, WO05/018638, WO05/019206,WO05/019205, WO05/018547, WO05/017190, US2005/0176776. in an embodimentinhibitors of mitotic kinesins include, but are not limited toinhibitors of KSP, inhibitors of MKLP1, inhibitors of CENP-E, inhibitorsof MCAK, inhibitors of Kif14, inhibitors of Mphosphl and inhibitors ofRab6-KIFL.

Examples of “histone deacetylase inhibitors” that can be used incombination with the compounds of the invention include, but are notlimited to, TSA, oxamflatin, PXD101, MG98, valproic acid and scriptaid.Further reference to other histone deacetylase inhibitors may be foundin the following manuscript; Miller, T. A. et al. J. Med. Chem.46(24_(.)):5097-5116 (2003).

Inhibitors of kinases involved in mitotic progression that can be usedin combination with the compounds of the invention include, but are notlimited to, inhibitors of aurora kinase, inhibitors of Polo-like kinases(PLK) (in particular inhibitors of PLK-1), inhibitors of bub-1 andinhibitors of bub-R1.

Antiproliferative agents that can be used in combination with thecompounds of the invention include, but are not limited to, antisenseRNA and DNA oligonucleotides such as G3139, ODN698, RVASKRAS, GEM231,and INX3001, and antimetabolites such as enocitabine, carmofur, tegafur,pentostatin, doxifluridine, trimetrexate, fludarabine, capecitabine,galocitabine, cytarribine ocfosfate, fosteabine sodium hydrate,raltitrexed, paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed,pernetrexed, neizarahine, 2′-deoxy-2′-methylidenecytidine,2′-fluommethylene-2′-deoxycytidine,N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl)urea,N6-[4-deoxy-4-[N2[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno-heptopyranosyl]adenine,aplidine, ecteinascidin, troxacitabine,4[2-amino-4-oxo-4,6,7,8-tarahydro-3H-pyrimidino[5,4-bi][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamicacid, aminopterin, 5-flurouracil, alanosine,11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetracyclo(7,4.1.0.0)-tetra.deca-2,4,6-trien-9-ylacetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase,2′-cyano-2′-deoxy-N4-palmitoyl-1-B-D-arabino furanosyl cytosine and3-aminopyridine-2-carboxablehyde thiosemicarbazone.

Examples of monoclonal antibody targeted therapeutic agents that. can beused in combination with the compounds of the invention include thosetherapeutic agents which have cytotoxic agents or radioisotopes attachedto a cancer cell specific or target cell specific monoclonal antibody,such as, for example, Bexxar.

Examples of HMG-CoA reductase inhibitors that may be used that can beused in combination with the compounds of the invention include, but arenot limited to, lovastatin (MEVACOR®; see U.S. Pat. Nos. 4,231,938,4,294,926 and 4,319,039), simvastatin (ZOCOR®; see U.S. Pat. Nos.4,444,784, 4,820,850 and 4,916,239), pravastatin (PRAVACHOL®; see U.S.Pat. Nos. 4,346,227, 4,537,859, 4,410,6:29, 5,030,447 and 5,180,589),fluvastatin (LESCOL®; see U.S. Pat, Nos. 5,354,772, 4,911,165,4,929,437, 5,189,164, 5,118,853, 5,290,946 and 5,356,896) andatorvastatin (LIPITOR®; see U.S. Pat. Nos. 5,273,995, 4,681,893,5,489,691 and 5,342,952). The structural formulas of these andadditional HMG-CoA reductase inhibitors that may be used in the instantmethods are. described at page 87 of M. Yalpani, “Cholesterol LoweringDrugs”, Chemistry & industry, pp. 85-89 (5 Feb. 1996) and U.S. Pat. Nos.4,782,084 and 4,885,314.

Examples of prenyl-protein transferase inhibitors that can be used incombination with the compounds of the invention include, but are notlimited to, can be found in the following publications and patents: WO96/30343, WO 97/18813, WO 97121701, WO 97/23478, WO 97/38665, WO98/28980, WO 98/29119, WO 95/32987, U.S. Pat. Nos. 5,420,245, 5,523,430,5,532,359, 5,510,510, 5,589,485, and 5,602,098, European Patent Publ. 0618 221, European Patent Publ. 0 675 112, European Patent Publ. 0 604181, European Patent Publ. 0 696 593, WO 94/19357, WO 95/08542, WO95/11917, WO 95/12612., WO 95/12572., WO 95/10514, U.S. Pat. No.5,661,152, WO 95/10515, WO 95/10516, WO 95/24612, WO 95/34535, WO95/25086, WO 96/05529, WO 96/06138, WO 96/06193, WO 96/16443, WO96/21701, WO 96/21456, WO 96/22278, WO 96/24611, WO 96/24612, WO96/05168, WO 96/05169, WO 96/00736, U.S. Pat. No. 5,571,792, WO96/17861, WO 96/33159, WO 96/34850, WO 96/34851, WO 96/30017, WO96/30018, WO 96/30362, WO 96/30363, WO 96/31111, WO 96/31477, WO96/31478, WO 96/31501, WO 97/00:252, WO 97/03047, WO 97/03050, WO97/04785, WO 97/02920, WO 97/17070, WO 97/23478, WO 97/26246, WO97/30053, WO 97/44350, WO 98/02436, and U.S. Pat. No. 5,532,359 For anexample of the role of a prenyl-protein transferase inhibitor onangiogenesis see European J. of Cancer, Vol. 35, No. 9, pp.1394-1401(1999).

Examples of angiogenesis inhibitors that can be used in combination withthe compounds of the invention include, but are not limited to, tyrosinekinase inhibitors, such as inhibitors of the tyrosine, kinase receptorsFit-1 (VEGFR1) and P1k-1/KDR (VEGFR2), inhibitors of epidermal-derived,fibroblast-derived, or platelet derived growth factors, MMP (matrixmetalloprotease) inhibitors, integrin blockers, interferon-a,interleukin-12, pentosan polysuifate, cyclooxygenase inhibitors,including nonsteroidal anti-inflammatories (NSAIDs) like aspirin andibuprofen as well as selective cyclooxy-genase-2 inhibitors likecelecoxib and rofecoxib (PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69,p. 475 (1982); Arch. Opthahnol., Vol, 108, p.573 (1990); Anat. Rec.,Vol. 238, p. 68 (1994); FEBS Letters, Vol. 372, p. 83 (1995); Clin,Orthop. Vol. 313, p. 76 (1995); J. Mol. Endocrinol., Vol. 16, p.107(1996); Jpn. J. Pharmacol., Vol. 75, p. 105 (1997); Cancer Res., Vol.57, p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Intl. Mol. Med., Vol.2, p. 715 (1998); J. Biol. Chem., Vol. 274, p. 9116 (1999), steroidalanti-inflammatories (such as corticosteroids, mineralocorticoids,dexamethasone, prednisone, prednisolone, methyipred, betamethasone),catboxyamidotriazole, combretastatin A-4, squalamine,6-O-chloroacetyl-cathonyl)-fumagillol, thalidomide, angiostatin,troponin-1, angiotensin II antagonists (see Fernandez et al., J. Lab,Clin. Med. 105:141-145 (1985)), and antibodies to VEGE (see, NatureBiotechnology, Vol. 17, pp.963-968 (October 1999); Kim et al., Nature,362, 841-844 (1993); WO 00/44777; and WO 00/61186).

Other therapeutic agents that modulate or inhibit angiogenesis may alsobe used in combination with the compounds of the instant invention andinclude agents that modulate or inhibit the coagulation and fibrinolysissystems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examplesof such agents that modulate or inhibit the coagulation and fibrinolysispathways that can be used in combination with the compounds of theinvention include, but are not limited to, heparin (see Thromb. Memos!.80:10-23 (1998)), low molecular weight heparins and carboxypeptidase Uinhibitors (also known as inhibitors of active thrombin activatablefibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354(2001)). TAFIa inhibitors have been described in PCT Publication WO03/013,526 and U.S. Ser. No. 60/349,925 (filed Jan. 18, 2002).

Agents that interfere with cell cycle checkpoints that can be used incombination with the compounds of the invention include, but are notlimited to, inhibitors of ATR, ATM, the Chk1 and Chk2 kinases and cdkand cdc kinase inhibitors and are specifically exemplified by7-hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.

Agents that interfere with receptor tyrosine kinases (RTKs) that can beused in combination with the compounds of the invention include, but arenot limited to, inhibitors of c-Kit, Eph, PDGF, Flt3 and FIBV. Furtheragents include inhibitors of RTKs as described by Bunce-Jensen andHunter, Nature, 411:355-365, 2001.

Inhibitors of cell proliferation and survival signaling pathway that canbe used in combination with the compounds of the invention include, butare not limited to, inhibitors of EGFR (for example gefitinib anderiotinib), inhibitors of ERB-2 (for example trastuzumab), inhibitors ofIGFR, inhibitors of cytokine receptors, inhibitors of HBV, inhibitors ofPI3K (for example LY294002), serine/threonine kinases (including but notlimited to inhibitors of Akt such as described in WO 02/083064, WO02/083139, WO 02/083140, US 2004-0116432, WO 02/083138, US 2004-0102360,WO 03/086404, WO 03/086279, WO 03/086394, WO 03/084473, WO 03/086403, WO2004/041162, WO 2004/096131, WO 2004/096129, WO 2004/096135, WO2004/096130, WO 2005/100356, WO 2005/100344), inhibitors of Raf kinase(for example BAY-43-9006), inhibitors of MEK (for example C1-1040 andPD-098059) and inhibitors of mTOR (for example Wyeth CCI-779). Suchagents include small molecule inhibitor compounds and antibodyantagonists.

Apoptosis inducing agents that can be used in combination with thecompounds of the invention include, but are not limited to, activatorsof TNF receptor family members (including the TRAIL receptors).

NSAIDs that are selective COX-2 inhibitors that can be used incombination with the compounds of the invention include, but are notlimited to, those NSAIDs disclosed in U.S. Pat. Nos. 5,474,995,5,861,419, 6,001,843, 6,020,343, 5,409,944, 5,436,265, 5,536,752.,5,550,142, 5,604,260, 5,698,584, and 5,710,140, WO 94/15932, U.S. Pat.Nos. 5,344,991, 5,134,142, 5,380,738, 5,393,790, 5,466,823, 5,633,272,and U.S. Pat. No. 5,932,598, all of which are hereby incorporated byreference.

Inhibitors of COX-2 that are particularly useful in combination with thecompounds of the invention include:3-phenyl-4-(4-(methylsulthnyl)phenyl)-2-(5H)-furanone; and5-chloro-3-(4-methylsulfonyl)-phenyl-2-(2-methyl-5-pyridinyl)pyridine;or a pharmaceutically acceptable salt thereof.

Compounds that have been described as specific inhibitors of COX-2 andare therefore useful in the present invention include, but are notlimited to: parecoxib, CELEBREX® and BEXTRA® or a pharmaceuticallyacceptable salt thereof.

Angiogenesis inhibitors that can be used in combination with thecompounds of the invention include, but are not limited to, endostatin,ukrain, ranpirnase, IM862,5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate,acetyldinanaline,5-amino-1-1-[[3,5-dichloro-4-(4-chlorobenzoyl)-phertyl]methyl]-1F1-1,2,3-triazole-4-carboxamide,CM101, squalamine, combretastatin, RPI4610,NX31838, sulfated mannopentaose phosphate,7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(1,3-naphthalenedisulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone(SU5416).

Tyrosine kinase inhibitors that can be used in combination with thecompounds of the invention include, but are not limited to,N-(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide,3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]indolin-2-one,17-(allylamino)-17-demethoxygeldanamycin,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline,N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine,BIBX1382,2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one,SH268, genistein, imatinib (STI571), CEP2563,4-(3-chlorophenylamino)-5,6-dimethyl.-7H-pyrrolo[2,3-d]pyrimidinemethanesulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline,4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquazoline, SU6668, STI571A, N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine, and. EMD121974.

Combinations with compounds other than anti-cancer compounds are alsoencompassed in the instant compositions and methods. For example,combinations of the instantly claimed compounds with PPAR-γ (Le.,PPAR-gamma) agonists and PPAR-δ (Le., PPAR-delta) agonists are useful inthe treatment of certain malignancies. PPAR-γ and PPAR-δ are the nuclearperoxisome proliferator-activated receptors γ and δ. The expression ofPPAR-γ on endothelial cells and its involvement in angiogenesis has beenreported in the literature (see J. Cardiovasc. Pharmacol. 31:909-913(1998); J. Biol. Chem. 274:9116-9121 (1999); Invest. Ophthalmol Vis.Sci. 41:2309-2317 (2000)). More recently, PPAR-γ agonists have beenshown to inhibit the angiogenic response to VEGE in vitro; bothtroglitazone and rosiglitazone maleate inhibit the development ofretinal neovascularization in mice. (Arch. Ophthamol. 119:709-717(2001)). Examples of PPAR-γ agonists and PPAR- γ/α agonists that can beused in combination with the compounds of the invention include, but arenot limited to, thiazolidinediones (such as DRF2725, CS-011,troglitazone, rosiglitazone, and pioglitazone), fenofibrate,gemfibrozil, clofibrate, GW2570, SB219994A.R-H039242, JTT-501, MCC-555,GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158, NN622, G1262570,PNU182716, DRF552926,2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropionicacid (disclosed in U.S. Ser. No. 09/782,856), and2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchromane-2-carboxylic acid (disclosed in U.S.Ser. Nos. 60/235,708 and 60/244,697).

Another embodiment of the instant invention is the use of the presentlydisclosed compounds in combination with gene therapy for the treatmentof cancer. For an overview of genetic strategies to treating cancer seeHall et al, (Am J Hum Genet 61:785-789 (1997)) and Kufe et al. (CancerMedicine, 5th Ed, pp 876-889, BC Decker, Hamilton, 2000). Gene therapycan be used to deliver any tumor suppressing gene. Examples of suchgenes include, but are not limited to, p53, which can be delivered viarecombinant virus-mediated gene transfer (see U.S. Pat. No. 6,069,134,for example), a uPA/uPAR antagonist (“Adenovirus-Mediated Delivery of auPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth andDissemination in Mice,” Gene Therapy, August 5(8):1105-13 (1998)), andinterferon gamma (J Immunol 164:217-222 (2000)).

The compounds of the instant invention may also be administered incombination with an inhibitor of inherent multidrug resistance (MDR), inparticular MDR associated with high levels of expression of transporterproteins. Such MDR inhibitors include inhibitors of p-glycoprotein(P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833(valspodar),

A compound of the present invention may be employed in conjunction withanti-emetic agents to treat nausea or emesis, including acute, delayed,late-phase, and anticipatory emesis, which may result from the use of acompound of the present invention, alone or with radiation therapy. Forthe prevention or treatment of emesis, a compound of the presentinvention may be used in conjunction with other and-emetic agents,especially neurokinin-1 receptor antagonists, 5HT3 receptor antagonists,such as ondansetron, granisetron, tropisetron, and zatisetron, GABABreceptor agonists, such as Baclofen, a corticosteroid such as Decadron(dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten orothers such as disclosed in U.S. Pat. Nos. 2,789,118, 2,990,401,3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326 and 3,749,712, anantidopaminergic, such as the phenothiazines (for exampleprochlorperazine, fluphenazine, thioridazine and mesoridazine),metoclopramide or dronabinol. In an embodiment, an anti-emesis agentselected from a neurokinin-1 receptor antagonist, a 5HT3 receptorantagonist and a corticosteroid is administered as an adjuvant for thetreatment. or prevention of emesis that may result upon administrationof the instant compounds.

Neurokinin-1 receptor antagonists of use in conjunction with thecompounds of the present invention are fully described, for example, inU.S. Pat. Nos. 5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595,5,459,270, 5,494.926, 5,496,833, 5,637,699, 5,719,147; European PatentPublication Nos. EP 0 360 390, 0 394 989, 0 428 434, 0 42.9 366, 0 430771, 0 436 334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901, 0512 902, 0 514 273, 0 514 274, 0 514 275, 0 514 276, 0 515 681, 0 517589, 0 520 555, 0 522 808, 0 528 495, 0 532 456, 0 533 280, 0 536 817, 0545 478, 0 558 156, 0 577 394, 0 585 913,0 590 152, 0 599 538, 0 610793, 0 634 402., 0 686 629, 0 693 489, 0 694 535, 0 699 655, 0 699 674,0 707 006, 0 708 101, 0 709 375, 0 709 376, 0 714 891, 0 723 959, 0 733632 and 0 776 893; PCT International Patent. Publication Nos. WO90/05525, 90/05729, 91/09844, 91/18899, 92/01688, 92/06079, 92/12151,92/15585, 92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93/00330,93/00331, 93/01159, 93/01165, 93/01169, 93/01170, 93/06099, 93/09116,93/10073, 93/14084, 93/14113, 93/18023, 93/19064, 93/21155, 93/21181,93/23380, 93/24465, 94/00440, 94/01402, 94/02461, 94/02595, 94/03429,94/03445, 94/04494, 94/04496, 94/05625, 94/07843, 94/08997, 94/10165,94/10167, 94/10168, 94/10170, 94/11368, 94/13639, 94/13663, 94/14767,94/15903, 94/19320, 94/19323, 94/20500, 94/26735, 94/26740, 94/29309,95/02595, 95/04040, 95/04042, 95/06645, 95/07886, 95/07908, 95/08549,95/11880, 95/14017, 95/15311, 95/16679, 95/17382, 95/18124, 95/18129,95/19344, 95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418,95/30674, 95/30687, 95/33744, 96/05181, 96/05193, 96/05203, 96/06094,96/07649, 96/10562, 96/16939, 96/18643, 96/20197, 96/21661, 96/29304,96/29317, 96/29326, 96/29328, 96/31214, 96/32385, 96/37489, 97/01553,97/01554, 97/03066, 97/08144, 97/14671, 97/17362, 97/18206, 97/19084,97/19942 and 97/21702; and in British Patent Publication Nos. 2 266 529,2 268 931, 2 269 170, 2 269 590, 2 271 774, 2 292 144, 2 293 168, 2 293169, and 2 302 689. The preparation of such compounds is fully describedin the aforementioned patents and publications, which are incorporatedherein by reference.

In an embodiment, the neurokinin- I receptor antagonist for use inconjunction with the compounds of the present invention is selectedfrom:2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)-phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)morpholine,or a pharmaceutically acceptable salt thereof, which is described inU.S. Pat. No. 5,719,147.

A compound of the instant invention may also be administered with anagent useful in the treatment of anemia. Such an anemia treatment agentis, for example, a continuous eythropoiesis receptor activator (such asepoetin alfa).

A compound of the instant invention may also be administered with anagent useful in the treatment of neutropenia. Such a neutropeniatreatment agent is, for example, a hematopoietic growth factor whichregulates the production and function of neutrophils such as a humangranulocyte colony stimulating factor, ((G-CSF). Examples of a G-CSFinclude filgrastim and PEG-filgrastim.

A compound of the instant invention may also be administered with animmunologic-enhancing drug, such as levamisole, isoprinosine andZadaxin.

A compound of the instant invention may also be useful for treating orpreventing liver disease or cancer in combination with other siNAtherapeutics.

The compounds of the instant invention may also be administered incombination with γ-secretase inhibitors and/or inhibitors of NOTCHsignaling. Such inhibitors include compounds described in WO 01/90084,WO 02/3091:2, WO 01/70677, WO 03/013506, WO 02/36555, WO 03/093252, WO03/093264, WO 03/093251, WO 03/093253, WO 2004/039800, WO 2004/039370,WO 2005/030731, WO 2005/014553, USSN 10/957,251, WO 2004/089911, WO02/081435, WO 02/081433, WO 03/018543, WO 2004/031137, WO 2004/031139,WO 2004/031138, WO 2004/101538, WO 2004/101539 and WO 02/47671(including LY-450139).

A compound of the instant invention may also be useful for treating orpreventing cancer in combination with PARP inhibitors.

A compound of the instant invention may also be useful for treatingcancer in combination with the following therapeutic agents: abarelix(Pienaxis depot®); aldesleukin (Prokine®); Aldesleukin (Proleukin®);Alemtuzurnabb (Campath®); alitretinoin (Panretin'®); allopurinol(Zyloprim®); altretamine (Hexalen®); amifostine (Ethyol®); anastrozole(Arimidex®); arsenic trioxide (Trisenox®); asparaginase (Elspar®);azacitidine (Vidaza®); bendamustine hydrochloride (Treanda®):bevacuzimab (Avastin®); bexarotene capsules (Targretin bexarotene gel(Targretin®); bleomycin (Blenoxane®); bortezomib (Velcade®); brefehlinA; busulfan intravenous (Busulfex®); busulfan oral (Myleran®);calusterone (Methosarb®); capecitabine (Xeloda®); carboplatin(Paraplatin®); carmustine (BCNU®, BiCNU®): carmustine (Gliadel®);carmustine with Polifeprosan 20 Implant (Gliadel Wafer®); celecoxib(Celebrex®); cetuximab (Erbitux®), chlorambucil (Leukeran®); cisplatin(Platinol®); cladrihine (Leustatin®, 2—CdA®); clofarahine (Clolar®);cyclophosphamide (Cytoxan®, Neosar®); cyclophosphamide (CytoxanInjection®); cyclophosphamide (Cytoxan Tablet®); cytarabine(Cytosar-U®); cytarabine liposomal (DepoCyt®); dacarbazine (DTIC-Dome®);dactinomycin, actinomycin D (Cosmegen®); &heparin sodium injection(Fraginin®); Darbepoefin alfa (Aranesp®); dasafinib (Sprycel®);daunorubicin liposomal (DanuoXome®); daunorubicin, daunomycin(Daunorubicin®); daunorubicin, daunomycin (Cerubidine®); degarelix(Firmagon®); Denileukin diftitox (Ontak®); dexrazoxane, (Zinecard®);dexrazoxane hydrochloride (Totect): didenmin B; 17-DMAG: docetaxel(Taxotere®); doxorubicin (Adriamycin PFS®); doxorubicin (Adriamycin®,Rubex®); doxorubicin (Adriamycin PFS injection®); doxorubicin liposomal(Doxil®); dromostanolone propionate (Dromostanolone, ®); dromostanolone,propionate (Masterone Injection®); eculizumab injection (Soliris®); BSolution (Elliott's B Solution®); eltrombopag (Promacta®); epirubicin(Ellence®); Epoetin alfa (epogen®); erlotinib (Tarceva®); estramustine(Emcyt®); ethinyl estradiol; etoposide phosphate (Etopophos®);etoposide, VP-16 (Vepesid®); everolimus tablets (Afinitor®); exemestane(Nromasin®); ferumoxytol (Feraheme Injection®); Filgrastim (Neupogen®);floxuridine (intraarterial) (FUDR®); fiudarabine (Fludara®);fluorouracil, 5-FU (Adrucli®); fulvestrant (Fasiodex®); gefitinib(Iressa®); geldanamycin; gemcitabine (Genizar®); gemtuzumab ozogamicin(Mylotarg®); goserelin acetate (Zoladex Implant®); goserelin acetate(Zoladex®); histrelin acetate (Ilistrelin implant®); hydroxyurea(Hydrea®); Ibritumomab Tiuxetan (Zevalin®); idarubicin (Idamycin®);ifosfamide (IFEX®); imatinib mesylate (Gleevec®); interferon alfa 2a(Roferon A®); Interferon alfa-2b (Intron AO); lobenguane I 123 injection(AdreView®); irinotecan (Camptosar®); ixabepilone (Lxempra®); lapatinibtablets (Tyketb®); lenalidomide (Revlimid®); letrozole (Femara®);leucovorin (Wellcovorin®, Leucovorin®); Leuprolide Acetate (Eligard®));levamisole (Ergamisol®); lomustine, CCNU (CeeBU®); meclorethamine,nitrogen mustard (Mustargen®); megestrol acetate (Megace®); melphalan,L-PAM (Alkeran®); mercaptopurine, 6-MP (Purinethol®); mesna (Mesnex®);mesna (Mesnex tabs®); methotrexate (Methotrexate®); methoxsalen(Uvadex®); 8-methoxypsoralert; mitomycin C (Mutamycin®); mitotane(Lysodren®); mitoxantrone (Novantrone®); mitramycin; nandrolonephenpropionate (Durabolin-50®); nelarabine (Arranon®); nilotinib(Tasigna®); Nofetumomab (Verluma®); ofatumumab (Arzerra®); Oprelvekin(Neuniega®); oxaliplatin (Eloxatin®); paclitaxel (Paxene®); paclitaxel(Taxol®); paclitaxel protein-bound particles (Abraxane®); palifermin(Kepivance®); pamidronate (Aredia®); panitumumab (Vectibix®); pazopanibtablets (Votrienttm®); pegademase (Adagen (Pegademase Bovine)®);pegaspargase (Oncaspar®); Pegfilgrastini (Neulasta®); pemetrexeddisodium (Alimta®); pentostatin (Nipent®); pipobroman (Vercyte®);pierixafor (Mozobil®); mithramycin (Mithracin®); porfimer sodium(Photofrin®); pralatrexate injection (Folotyn®); procarbazine(Matulane®); quinacrine (Atabrine®); rapamycin; Rasburicase (Elitek®);raloxifene hydrochloride (Evista®); Rituximab (Rituxan®); romidepsin(Istodax®); romiplostim (Nplate®); sargramostim (Leukine®); Sargramostim(Prokine®); sorafenib (Nexavar®); streptozocin (Zanosar®); sunitinibmaleate (Sutent®); talc (Scierosol®); tamoxifen (Nolvadex®);temozolomide (Temodar®); temsirolimus (Torisel®); teniposide, VM-26(Vumon®); testolactone (Teslac®); thioguanine, 6-TG (Thioguanine®);thiopurine; thiotepa (Thioplex®); topotecan (Hycamtin®); toremifene(Fareston®); Tositurnomab (Bexxar®); Tositamomab/I-131 tositumomab(Bexxar®); trans-retinoic acid; Trastuzumab (Herceptin®); tretinoin,ATRA (Vesanoid®); triethylenemelamine; Uracil Mustard (Uracil MustardCapsules®); valrubicin (Valstar®) vinblastine (Velban®); vincristine(Oncovin®) vinorelbine (Navelbine®); vorirtostat (Zolinza®); wortmannin;and zoledronate (Zonieta®).

The invention also provides a combination comprising an siNA molecule ofthe invention targeting one gene together with another inhibitortargeting a second target gene.

The combinations referred to above can conveniently be presented for usein the form of a pharmaceutical formulation and thus pharmaceuticalcompositions comprising a combination as defined above together with apharmaceutically acceptable diluent or carrier represent a furtheraspect of the invention.

To practice the coordinate administration methods of this disclosure, ansiNA molecule is administered simultaneously or sequentially in acoordinated treatment protocol with one or more secondary or adjunctivetherapeutic agents described herein or known in the alt The coordinateadministration may be done in either order, and there may be a timeperiod while only one or both (or all) active therapeutic agents,individually or collectively, exert their biological activities. Adistinguishing aspect of all such coordinate treatment methods is thatthe siNA molecule(s) present in a composition elicits some favorableclinical response, which may or may not be in conjunction with asecondary clinical response provided by the secondary therapeutic agent.For example, the coordinate administration of an siNA molecule with asecondary therapeutic agent as contemplated herein can yield an enhanced(e.g., synergistic) therapeutic response beyond the therapeutic responseelicited by either or both the purified siNA molecule and the secondarytherapeutic agent alone.

The individual compounds of such combinations can be administered eithersequentially or simultaneously in separate or combined pharmaceuticalformulations. in one embodiment, the individual compounds will beadministered simultaneously in a combined pharmaceutical formulation.

Thus, the described molecules could be used in combination with one ormore known compounds, treatments, or procedures to prevent or treatdiseases, disorders, conditions, and traits described herein in asubject or organism as are known in the art, such as other geneinhibitors.

3, Therapeutic Applications

The present body of knowledge in RNAi. research indicates the need formethods that can modulate gene expression for therapeutic use.

Thus, one aspect of the invention comprises a method of treating asubject including, but not limited to, a human suffering from a diseaseor a condition which is mediated by the action of target geneexpression, which method comprises administering to said subject aneffective amount of a double-stranded siNA molecule of the invention. Inone embodiment of this aspect, the siNA molecules comprises sequencehaving at least a 15 nucleotides complementary to a target nucleic acid.In other embodiments, the siNA molecule comprises any molecule hereinhaving formula (A).

In some embodiments of this aspect, the disease or condition is cancer,a proliferative, inflammatory, autoimmune, neurologic, ocular,respiratory, metabolic, dermatological, auditory, liver, kidney, orinfectious disease as described herein or otherwise known in the art.Thus, in certain embodiments the molecules and compositions of theinstant invention are useful in a method for treating cancer,proliferative, inflammatory, autoimmune, neurologic, ocular,respiratory, metabolic, dermatological, auditory, liver, kidney, orinfectious diseases.

In certain embodiments, the administration of the siNA molecule is vialocal administration or systemic administration. In other embodiments,the invention features contacting the subject or organism with an siNAmolecule of the invention via local administration to relevant tissuesor cells, such as lung cells and tissues, such as via pulmonarydelivery. In yet other embodiments, the invention features contactingthe subject or organism with an siNA molecule of the invention viasystemic administration (such as via intravenous or subcutaneousadministration of siNA) to relevant tissues or cells in a subject ororganism.

siNA molecules of the invention are also used as reagents in ex vivoapplications. For example, siNA reagents are introduced into tissue orcells that are transplanted into a subject for therapeutic effect. Thecells and/or tissue can be derived from an organism or subject thatlater receives the explant, or can be derived from another organism orsubject prior to transplantation. The siNA molecules can be used tomodulate the expression of one or more genes in the cells or tissue,such that the cells or tissue obtain a desired phenotype or are able toperform a function when transplanted in vivo. In one embodiment, certaintarget cells from a patient are extracted. These extracted cells arecontacted with siNAs targeting a specific nucleotide sequence within thecells under conditions suitable for uptake of the siNAs by these cells(e.g., using delivery reagents such as cationic lipids, liposomes andthe like or using techniques such as electroporation to facilitate thedelivery of siNAs into cells). The cells are then reintroduced back intothe same patient or other patients.

For therapeutic applications, a pharmaceutically effective dose of thesiNA molecules or pharmaceutical compositions of the invention isadministered to the subject. A pharmaceutically effective dose is thatdose required to prevent, inhibit the occurrence, or treat (alleviate asymptom to some extent, preferably all of the symptoms) a disease state.One skilled in the art can readily determine a therapeutically effectivedose of the siNA of the invention to be administered to a given subject,by taking into account factors, such as the size and weight of thesubject, the extent of the disease progression or penetration, the age,health, and sex of the subject, the route of administration, and whetherthe administration is regional or systemic. Generally, an amount between0.1 μg/kg and 140 mg/kg body weight/day of active ingredients isadministered dependent upon potency of the siNA of the disclosure. Theamount of active ingredient that can be combined with the carriermaterials to produce a single dosage form varies depending upon the hosttreated and the particular mode of administration. Optimal dosingschedules can be calculated from measurements of drug accumulation inthe body of the patient. The siNA molecules of the invention can beadministered in a single dose or in multiple doses.

siNA molecules of the instant invention can be administered oncemonthly, once weekly, once daily (QD), or divided into multiple monthly,weekly, or daily doses, such as, for example, but not limitation, twicedaily (BID), three times daily (T1D), once every two weeks. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues.

In addition, the administration can be continuous, i.e., every day, orintermittently. For example, intermittent administration of a compoundof the instant invention may be administration one to six days per weekor it may mean administration in cycles (e.g. daily administration fortwo to eight consecutive weeks, then a rest period with noadministration for up to one week) or it may mean administration onalternate days.

G. Administration

Compositions or formulations can be administered in a variety of ways.Non-limiting examples of administration methods of the invention includeoral, buccal, sublingual, parenteral (i.e., intraarticularly,intravenously, intraperitoneally, subcutaneously, or intramuscularly),local rectal administration or other local administration. In oneembodiment, the composition of the invention can be administered byinsufflation and inhalation. Administration can be accomplished viasingle or divided doses. In some embodiments, the pharmaceuticalcompositions are administered intravenously or intraperitoneally by abolus injection (see, e.g., U.S, Pat. No. 5,286,634),

siNA molecule with or without a vehicle can be locally delivered bydirect injection or by use of an infusion pump. Direct injection of thesiNA molecules of this disclosure, whether subcutaneous, intramuscular,or intradermal, can take place using standard needle and syringemethodologies, or by needle free technologies, such as those describedin Conroy et al, (1999, Clin. Cancer Res. 5:2330) and PCT PublicationNo. WO 99/31262. For example, but not limitation, lipid nucleic acidparticles can be administered by direct injection at the site of diseaseor by injection at a site distal from the site of disease (see, e.g.,Culver, HUMAN GENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York.pp. 70-71(1994)). In one embodiment, the siNA molecules of the inventionand formulations or compositions thereof are administered to a cell,subject, or organism as is described herein and as is generally known inthe art,

1. In Vivo Administration

In any of the methods of treatment of the invention, the siNA can beadministered to the subject systemically as described herein orotherwise known in the art, either alone as a monotherapy or incombination with additional therapies described herein or as are knownin the art. Systemic administration can include, for example, pulmonary(inhalation, nebulization etc,) intravenous, subcutaneous,intramuscular, catheterization, nasopharyngeal, transdermal, ororal/gastrointestinal administration as is generally known in the art.

In any of the methods of treatment or prevention of the invention, thesiNA can be administered to the subject locally or to local tissues asdescribed herein or otherwise known in the art, either alone as amonotherapy or in combination with additional therapies as are known inthe art. Local administration can include, for example, inhalation,nebulization, catheterization, implantation, direct injection,dermal/transdermal application, patches, stenting, ear/eye drops, orportal vein administration to relevant tissues, or any other localadministration technique, method or procedure, as is generally known inthe art.

In one embodiment, the siNA molecules of the invention and formulationsor compositions thereof are administered to the liver as is generallyknown in the art (see for example Wen et al., 2004, World JGastroenterol., 10, 244-9; Murao et al., 2002, Pharm Res., 19, 1808-14;Liu et al., 2003, gene Ther., 10, 180-7; Hong et al., 2003, J PharmPharmacol., 54, 51-8; Herrmann et al., 2004, Arch Virol., 149, 1611-7;and Matsuno et al., 2003, gene Ther., 10, 1559-66).

In one embodiment, the invention features the use of methods to deliverthe siNA molecules of the instant invention to hematopoietic cells,including monocytes and lymphocytes. These methods are described indetail by Hartmann et al., 1998, J. Phamacol, Exp. Ther., 285(2),920-928; Kronenwett et al., 1998, Blood, 91(3), 852-862; Filion andPhillips, 1997, Biochim. Biophys. Acta., 1329(2), 345-356; Ma and Wei,1996, Leuk. Res., 20(11/12), 925-930; and Bongartz et al., 1994, NucleicAcids Research, 22(22), 4681-8.

In one embodiment, the siNA molecules of the invention and formulationsor compositions thereof are administered directly or topically (e.g.,locally) to the dermis or follicles as is generally known in the art(see for example Brand, 2001, Curr. Opin. Mol. Ther., 3, 244-8; Regnieret 01., 1998, J. Drug Target, 5, 275-89; Kanikkannan, 2002, BioDrugs,16, 339-47; Wraight et al., 2001, Pharmacol. Ther., 90, 89-104; andTreat and Dujardin, 2001, STP PharmaSciences, 11, 57-68). In oneembodiment, the siNA molecules of the invention and formulations orcompositions thereof are administered directly or topically using ahydroalcoholic gel formulation comprising an alcohol (e.g., ethanol orisopropanol), water, and optionally including additional agents suchisopropyl myristate, and carbomer 980. In other embodiments, the siNAare formulated to be administered topically to the nasal cavity. Topicalpreparations can be administered by one or more applications per day tothe affected area; over skin areas occlusive dressings canadvantageously be used. Continuous or prolonged delivery can be achievedby an adhesive reservoir system.

In one embodiment, an siNA molecule of the invention is administerediontophoretically, for example to a particular organ or compartment(e.g., the eye, back of the eye, heart, liver, kidney, bladder,prostate, tumor, CNS etc,). Non-limiting examples of iontophoreticdelivery are described in, for example, WO 03/043689 and WO 03/030989,which are incorporated by reference in their entireties herein.

In one embodiment, the siNA molecules of the invention and formulationsor compositions thereof are administered to the lung as is describedherein and as is generally known in the art. In another embodiment, thesiNA molecules of the invention and formulations or compositions thereofare administered to lung tissues and cells as is described in U.S.Patent Publication Nos. 2006/0062758; 2006/0014289; and 2004/0077540.

2. Aerosols and Delivery Devices

a. Aerosol Formulations

The compositions of the present invention, either alone or incombination with other suitable components, can be made into aerosolformulations (i.e., they can be “nebulized”) to be administered viainhalation (e.g., intranasally or intratracheally) (see, Brigham et al.,Am, J. Sci., 298:278 (1989)). Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

In one embodiment, the siNA molecules of the invention and formulationsthereof are administered via pulmonary delivery, such as by inhalationof an aerosol or spray dried formulation administered by an inhalationdevice or nebulizer, providing rapid local uptake of the nucleic acidmolecules into relevant pulmonary tissues. Solid particulatecompositions containing respirable dry particles of micronized nucleicacid compositions can be prepared by grinding dried or lyophilizednucleic acid compositions, and then passing the micronized compositionthrough, for example, a 400 mesh screen to break up or separate outlarge agglomerates. A solid particulate composition comprising the siNAcompositions of the invention can optionally contain a dispersant whichserves to facilitate the formation of an aerosol as well as othertherapeutic compounds. A suitable dispersant is lactose, which can beblended with the nucleic acid compound in any suitable ratio, such as a1 to 1 ratio by weight.

Spray compositions comprising siNA molecules or compositions of theinvention can, for example, be formulated as aqueous solutions orsuspensions or as aerosols delivered from pressurized packs, such as ametered dose inhaler, with the use of a suitable liquefied propellant.in one embodiment, aerosol compositions of the invention suitable forinhalation can be either a suspension or a solution and generallycontain an siNA molecule comprising formula (A), and a suitablepropellant such as a fluorocarbon or hydrogen-containingchlorofluorocarbon or mixtures thereof, particularlyhydrofitioroalkanes, especially 1,1,1,2-tetrafluoroethane,1,1,1,2,3,3,3-heptafluoro-n-propane or a mixture thereof. The aerosolcomposition can optionally contain additional formulation excipientswell known in the art such as surfactants. Non-limiting examples includeoleic acid, lecithin or an oligolactic acid or derivative such as thosedescribed in WO94/21229 and WO98/34596 and co-solvents for exampleethanol. In one embodiment a pharmaceutical aerosol formulation of theinvention comprising a compound of the invention and a fluorocarbon orhydrogen-containing chlorofluorocarbon or mixtures thereof aspropellant, optionally in combination with a surfactant and/or aco-solvent.

The aerosol formulations of the invention can be buffered by theaddition of suitable buffering agents.

Aerosol formulations can include optional additives includingpreservatives if the formulation is not prepared sterile. Non-limitingexamples include, methyl hydroxybenzoate, anti-oxidants, flavorings,volatile oils, buffering agents and emulsifiers and other formulationsurfactants. In one embodiment, fluorocarbon or perfluorocarbon carriersare used to reduce degradation and provide safer biocompatiblenon-liquid particulate suspension compositions of the invention (e.g.,siNA and/or LNP formulations thereof). In another embodiment, a devicecomprising a nebulizer delivers a composition of the invention (e.g.,siNA and/or LNP formulations thereof) comprising fluorochemicals thatare bacteriostatic thereby decreasing the potential for microbial growthin compatible devices.

Capsules and cartridges comprising the composition of the invention foruse in an inhaler or insufflator, of for example gelatin, can beformulated containing a powder mix for inhalation of a compound of theinvention and a suitable powder base such as lactose or starch. In oneembodiment, each capsule or cartridge contains an siNA moleculecomprising formula (A), and one or more excipients. In anotherembodiment, the compound of the invention can be presented withoutexcipients such as lactose

The aerosol compositions of the present invention can be administeredinto the respiratory system as a formulation including particles ofrespirable size, particles of a size sufficiently small to pass throughthe nose, mouth and larynx upon inhalation and through the bronchi andalveoli of the lungs. In general, respirable particles range from about0,5 to 10 microns in size. In one embodiment, the particulate range canbe from 1 to 5 microns. In another embodiment, the particulate range canbe from 2 to 3 microns. Particles of non-respirable size which areincluded in the aerosol tend to deposit in the throat and be swallowed,and the quantity of non-respirable particles in the aerosol is thusminimized For nasal administration, a particle size in the range of10-500 um is preferred to ensure retention in the nasal cavity.

In some embodiments, an siNA composition of the invention isadministered topically to the nose for example, for the treatment ofrhinitis, via pressurized aerosol formulations, aqueous formulationsadministered to the nose by pressurized pump or by nebulization.Suitable formulations contain water as the diluent or carrier for thispurpose. In certain embodiments, the aqueous formulations foradministration of the composition of the invention to the lung or nosecan be provided with conventional excipients such as buffering agents,tonicity modifying agents and the like.

b. Devices

The siNA molecules of the invention can be formulated and delivered asparticles and/or aerosols as discussed above and dispensed from variousaerosolization devices known by those of skill in the art.

Aerosols of liquid or non-liquid particles comprising an siNA moleculeor formulation of the invention can be produced by any suitable means,such as with a device comprising a nebulizer (see for example U.S. Pat.No. 4,501,729) such as ultrasonic or air jet nebulizers.

Solid particle aerosols comprising an siNA molecule or formulation ofthe invention and surfactant can be produced with any solid particulateaerosol generator. One type of solid particle aerosol generator usedwith the siNA molecules of the invention is an insufflator. A secondtype of illustrative aerosol generator comprises a metered dose inhaler(“MDI”). MDIs containing siNA molecules or formulations taught hereincan be prepared by methods of the art (for example, see Byron, above andWO96/32099).

The siNA molecules can also be formulated as a fluid formulation fordelivery from a fluid dispenser, such as those described and illustratedin WO05/044354.

In certain embodiments of the invention, nebulizer devices are used inapplications for conscious, spontaneously breathing subjects, and forcontrolled ventilated subjects of all ages. The nebulizer devices can beused for targeted topical and systemic drug delivery to the lung. In oneembodiment, a device comprising a nebulizer is used to deliver an siNAmolecule or formulation of the invention locally to lung or pulmonarytissues. In another embodiment, a device comprising a nebulizer is usedto deliver a an siNA molecule or formulation of the inventionsystemically.

H. Other Applications/Uses siNA Molecules of the Invention

The siNA molecules of the invention can also be used for diagnosticapplications, research applications, and/or manufacture of medicants,

In one aspect, the invention features a method for diagnosing a disease,trait, or condition in a subject comprising administering to the subjecta composition of the invention under conditions suitable for thediagnosis of the disease, trait, or condition in the subject.

In one embodiment, siNA molecules of the invention are used to downregulate or inhibit the expression of proteins arising from haplotypepolymorphisms that are associated with a trait, disease or condition ina subject or organism. Analysis of genes, or protein or RNA levels canbe used to identify subjects with such polymorphisms or those subjectswho are at risk of developing traits, conditions, or diseases describedherein. These subjects are amenable to treatment, for example, treatmentwith siNA molecules of the invention and any other composition useful intreating diseases related to target gene expression. As such, analysisof protein or RNA levels can be used to determine treatment type and thecourse of therapy in treating a subject. Monitoring of protein or RNAlevels can be used to predict treatment outcome and to determine theefficacy of compounds and compositions that modulate the level and/oractivity of certain peptides and/or proteins associated with a trait,disorder, condition, or disease.

In another embodiment, the invention comprises use of a double-strandednucleic acid according to the invention for use in the manufacture of amedicament. In an embodiment, the medicament is for use in treating adisease or a condition that is mediated by the action of one or moretarget genes. In one embodiment, the medicant is for use in treating anydisease or condition herein or otherwise known in the art. In someembodiments, the medicament is for use in the treatment of cancer.

In certain embodiments, the siNA molecules of the invention are for usein a method for treating any disease or condition contemplated herein.

In certain embodiments, the siNA molecules of the invention are for usein a method for treating cancer.

I. EXAMPLES

The invention will now be illustrated with the following non-limitingexamples. Those of skill in the art will readily recognize a variety ofnon-critical parameters which can be changed or modified to yieldessentially the same results.

Example 1 Identification of Highly Potent Stabilized siNA Molecules withProlonged Duration

The development of therapeutic siRNAs with drug-like properties isdependent on the incorporation of chemical modifications to improveduration of RNA knockdown and potency while minimizing non-specific(off-target) effects and stimulation of innate immunity. The growingdiversity of siRNA delivery vehicles has expanded beyond traditionallipid encapsulated nano-particles to include dynamic polymer conjugatesand conjugation with various targeting ligands including antibodies,sugars, and cholesterol. Many of these delivery strategies expose thesiRNA cargo to serum and/or cellular nucleases which can compromise thestructural integrity and in vivo efficacy of both unmodified siRNAs andmodified siRNAs which have not been optimized for nuclease stability.Additionally, these alternative delivery strategies employ differenttargeting ligands and mechanisms of endosomal escape, thereby exposingthe siRNA cargo to different cellular micro-environments with varied pHand nuclease content. Therefore optimization of siRNA stability isneeded for development of therapeutic siRNAs with cross-deliveryplatform compatibility and potentially enhanced duration due toimprovement of stability attributes along with pharmacokinetic andpharmacodynamic profiles of the siRNA molecule.

The modified siNA molecules disclosed herein (see for example sequencesdescribed below with reference to particular sequences in Table 1) areshown to significantly improve serum stability while maintaining robustpotency. The modification criteria of the present invention can beapplied to any siRNA sequence. Furthermore, the presence of2′-deoxy-2′-fluoro modifications, and in particular 2′-deoxy-2′-fluoropurine modifications in conjunction with 2′-O-methyl pyrimidinemodifications, are shown to be important for optimal siRNA duration ofknockdown in vivo.

Traditional lipid nanoparticle (LNP) delivery vehicles encapsulate thesiRNA and thus limit exposure to serum nucleases upon intravenous dosingof siRNA-LNP complexes in animals. The adoption of alternative deliveryplatforms such as polymer conjugates (PC) or direct attachment ofdelivery targeting ligands (e.g., cholesterol) exposes the siRNAmolecule to serum nucleases and potentially hostile intracellularenvironments. Therefore the. development of a stabilized siRNAs withstrategies that can be applied to siRNA irrespective of sequence toimprove nuclease/chemical stability while retaining requisite RNAknockdown and potency is useful in conjunction with heterogeneousdelivery vehicle platforms.

The Sci10 Modification Motif

The Stab 07/35 modification motif (see Table 8) is a combination of2′-deoxy (2′H) purines and 2′-deoxy-2′-fluoro (2′F) pyrimidines on thepassenger strand and 2′-O-methyl (2′-OMe) purines and 2′-deoxy-2′-fluoro(2′F) purines on the guide strand. When applied to an ApoB (9514) siRNA,the 07/35 motif was shown to have high passenger strand serum stabilityhowever the guide strand was susceptible to nuclease degradation (seeFIG. 11) in serum. The addition of phosphorothioate linkages at the 3′ends of both strands (07H/35N motif) improved stability. Additionalphosphorothioate modification to the 5′-end of the guide strand (motif07H/35U2) further improved stability. The RNASci10 modification motifrepresents a departure from the 07/35 modification motif in whichpurines are 2′F modified and pyrimidines are 2′OMe modified, andposition 14 (counting from the 5′-end of the guide strand) of the guidestrand is a 2′F nucleotide regardless of underlying purine or pyrimidineidentity (see FIG. 11). The Sci10 modification motif shows improvedpassenger and guide strand stability while retaining in vitro mRNAknockdown and potencies comparable to the 07/35 motif (see FIG. 11 andTable 2).

Position 14 effects

Position 14 of the guide strand can be sensitive to particular 2′-ribosesugar modifications. An evaluation of 2′1=′, 2′OMe and 2′H modificationof this position was conducted on 7 otherwise unmodified siRNA sequences(see FIG. 12), 2′OMe content at this position was poorly tolerated while2′H was reasonably tolerated relative to siRNAs unmodified at thisposition (i.e. 2′OH at position 14). However 2′F was well tolerated andeven slightly improved mRNA knockdown was observed relative tounmodified. Therefore, a preferred embodiment of the present inventionfeatures a 2′F moiety at position 14 of the guide strand (regardless ofunderlying pyrimidine purine identity) of any siNA molecule of theinvention (e.g., a siNA molecule having Formula A herein or as describedin Table 8).

The Apori siRNAs described in FIG. 11 were covalently attached to thepolymer conjugate via a disulfide. linker on the 5′ end of the passengerstrand and tested for mRNA knockdown in mouse livers in vivo. At day 2of the in vivo study, ApoB mRNA knockdown by polymer conjugate (PC)Sci10 exceed that measured for the 07/35 motifs tested (FIG. 13A-B,Table 2). The separation of Sci10 from 07/35 chemistries extended at day7 of the in vivo studies, demonstrating robust duration of mRNAknockdown out to 21 days (FIG. 13B, Table 2).

The Sci1.0 modification chemistry was also effective when delivered bylipid nanoparticle (LNP) (FIGS. 14A-14B, Table 3) formulations. A thirdindependent in vivo study with PC delivered siRNAs demonstrated certainadvantages of the Sci10 motif versus the 07/35 motif (FIG. 14A).However, when delivered with LNP the 07/35 motif is equally effective toSci10. This can most likely be attributed to the encapsulation of siRNAcargo and protection from serum nucleases noted for the LNP deliveryplatforms. Also of note is the delivery vehicle-dependent differences induration of mRNA knockdown at day 7 of the in vivo studies. The PCdelivered Sci10 construct is 4-fold more active than either of the LNPdelivered siRNAs at the same day 7 time point.

The incorporation of phosphorothioates at positions 1-3 of the guidestrand can improve the stability of the guide strand, see for example07H/35N and 07H/35U2 (FIG. 11). However, each phosphorothioateincorporation can generate mixtures of chiral products and thephosphorothioate modification can be sensitive to oxidative reversionback to a phosphodiester linkage. Therefore an identified set of 2′ribose modifications to positions 1-3 of the guide strand were evaluatedfor their in vitro stability and mRNA knockdown/potency (FIG. 15 andTable 4). These four modification motifs to the 5′ of the guide strandare tolerated, retaining RNA knockdown levels, and have stabilityprofiles equivalent to the phosphorothioates they replace. Thereforethese 5′-guide strand 2′ ribose modification motifs are preferredbecause of their ability to replace phosphorothioates while retainingbeneficial stability properties. This extends in vivo where the fouridentified guide strand 5′-end modification motifs have equivalentlevels of mRNA knockdown and duration (FIG. 16 and Table 4).

After demonstrating the value of the Sci10 modification motif for ApoB(9514) another siRNA was selected to evaluate whether the Sci10modification motif could be applied to multiple siRNAs of varyingsequence. FIG. 17 shows the in vitro mRNA knockdown and serum stabilityfor a series of SSB (291) siRNAs. As with ApoB, the 07/35-basedmodification motif had less serum stability. However, the Sci10modification motif containing phosphorothioates at the 5′-end of theguide was surprising unstable. An inspection of the nucleotide sequenceof the 5′ guide strand identified a “UA” motif at positions 2-3. Thissequence motif is susceptible to nuclease cleavage activity. Forexample, known sequence preferences of RNase-like activities on RNAtemplates include UA, CA, UG, and CG motifs that are susceptible tonuclease. Because pyrimidines preceding an adenosine residue appear tobe most susceptible, this motif is noted as “YA” in FIG. 17 with “Y”representing pyrimidines. Interestingly, the application of the 2′ribose modification motifs discussed for ApoB (FIG. 15) conferred astriking improvement in the guide strand serum stability of thesesiRNAs. This observation highlights another liability of usingphosphorothioates for serum stability in the context of nucleasecleavage hotspots phosphorothioates are less effective while 2′ ribosemodifications confer robust nuclease stability (Table 5). Two of theSci10 modification motifs (ScilOdffn and Sci10ffd) were selected for invivo testing (FIG. 18). Compared to the 07/35 modification motif, thetwo Sci10 with specific 5′-end modification motif selections possesssignificant RNA knockdown that extends out to 3 weeks in vivo (FIG. 18and Table 5).

Given the advantageous properties of the Sci10 modification motif,including improved nuclease stability, significant in vivo duration ofknockdown and retention of siRNA potency; applicant chose to evaluatethe contribution of 2′F purine modifications for ApoB (9514). The Sci11modification motif removes the 2′F purine modifications from thepassenger strand, replacing with 2′OH, while the Sci07f modificationmotif removes the 2′F purine modification content from both passengerand guide strands. Note that 2′F modification at position 14 of theguide strand is retained. In vitro mRNA knockdown and serum stabilitywas observed to be nearly equivalent among these siRNAs, although theSci11 motif demonstrated slightly reduced serum stability (FIG. 19A).However, when assessed in vivo, there was a significant and strikingseparation of these siRNAs (FIG. 19B) in terms of activity. The Sci10and Sci11 motifs demonstrated equivalent knockdown at day 2 while theSci07f motif was surprisingly compromised in comparison. As themeasurement of duration of ApoB RNA knockdown continued for day 7, 14,and 21 time points, it become clear that the Sci1.0 motif has superiorduration (FIG. 19B and Table 6). Taken together these data suggest thatthe 2′F purine content on the passenger and guide strand of the Sci10motif is conferring an advantageous property. To investigate this, thelivers from siRNA treated mice were homogenized and mass spectrometrywas used to evaluate the in vivo metabolism of the PC-delivered siRNAs(FIG. 19C). The Sci 10 siRNA was found to only have minor sites ofmetabolism—defined as less than 10% of parental strand at 48 hours. When2′F modified purines are removed from the passenger strand (Sci11) thereis a resulting increase in major sites of metabolism on both thepassenger and guide strands. Guide strand metabolism can be attributedto loss of the nuclease protective nature of an intact duplex. in otherwords, a labile passenger strand can exposes the guide strand tonuclease degradation. When 2′F purines are removed from both passengerand guide strands (Sci.070, the in vivo stability of the siRNA isfurther compromised.

A similar evaluation of 2′F content was extended to SSB (291) siRNAs.The 2′F purines of Sci10dfm and Sci10ffd motifs (see FIGS. 17 and 18)were replaced with 2′OH resulting in Sci07dfm and Sci07ffd siRNAs (FIG.20A). As with ApoB, the in vitro mRNA knockdown and stability of theSci07 and Sci10 siRNAs was largely comparable. However, in vivo therewas a marked difference in mRNA knockdown and duration between these twomotifs (FIG. 20B). The 2′F purine containing Sci10 siRNAs possessedsuperior initial knockdown which persisted at least 3 weeks in vivowhereas the Sci07 siRNAs without 2′F content had poor initial knockdownand limited duration (Table 7). The SSB (291) recapitulation of theobserved ApoB dependence on 2′F content, specifically 2′F purine incombination with 2′OMe pyrimidine modifications, suggests that 2′Fcontent and it's contribution to in vivo duration is a generalphenomenon.

A follow-up study evaluated the tolerance of the Sci 10 modificationmotif across 80 different ApoB, SSB, and PHD2 sequences. The Sci10 motifwas applied to multiple different target sequences and in vitroknockdown activity was assessed as compared to corresponding minimallymodified controls. The minimal modification motif (09H/10N) hasribonucleotides (2′OH) at all positions of the duplex with 2′-OMeuridine overhangs with a single thioate residue between the 2′OMe-U's atthe 3′ end of each strand. By comparing in vitro knockdown of twodifferent siRNAs (minimally modified 09H-10N versus Sci10) correspondingto the same sequence, the impact of Sci10 modification was assessed on awide variety of different siRNA sequences. This analysis includes 29different ApoB sequences, 24 different PHD2 sequences, and 27 differentSSB sequences (see Tables 10, 11, and 12 respectively). Knockdown oftarget mRNA was measured at 10 nM and 1 nM concentrations to determinethe impact of Sci10 modification on the potency of the tested siRNAs.Comparison of 09H/10N and Sci10 was performed on a pair-wise basis foreach of the 80 different siRNA sequences. The difference in knockdown(in log 2) was calculated by subtracting 09H/10N knockdown levels fromthose measured for the Sci10 motif. Positive values indicate the Sci10motif is more active than minimally modified 09H/10N; an unexpectedresult for highly modified siRNAs. Negative values indicate that theSci10 modification was less active relative to 09H/10N. The experimentalvariation and accuracy of the qPCR assay is approximately 0.5 (log 2).Therefore values within 0.5 of the 09H/10N are considered to beequivalent in overall knockdown and therefore equally tolerated. Thisdata is summarized in Table 13.

In conclusion, incorporation of 2′OMe pyrimidine and 2′F purinemodifications in the passenger and guide strands of siRNAs are shown todemonstrate improved siRNA serum stability. This RNASci10 modificationmotif improves siRNA stability while retaining potency of RNA knockdown.RNASci10 provides compatibility with RNA delivery vehicles such aspolymer conjugates and ligand conjugates where the siRNA is exposed toserum and/or hostile cellular environments. Additionally, thismodification motif improves duration of siRNA mediated RNA knockdown invivo via a mechanism that appears to benefit from the presence of 2′Fmodifications, specifically 2′F purine combined with 2′OMe pyrimidinemodifications. The combination of the RNASci10 modification motif withindependently identified modifications to positions 1-3 of the 5′-end ofthe guide strand results in siRNAs which are 100% modified and possesshigh stability, potency, and in vivo duration. Because of their inherentnuclease stability, siRNAs containing these modification motifs can becoupled with cell or tissue targeting ligands (e,g. antibodies, sugarsor cholesterol) to create effective self-delivering siRNAs.

MATERIALS AND METHODS

siNA Synthesis

For each oligonucleotide of a target sequence, the two individual,complementary strands of the siNA were synthesized separately usingsolid phase synthesis, then purified separately by reversed phase solidphase extraction (SPE). The complementary strands were annealed to formthe double strand (duplex) and delivered in the desired concentrationand buffer of choice.

Briefly, the single strand oligonucleotides were synthesized usingphosphoramidite chemistry on an automated solid-phase synthesizer, usingprocedures as are generally known in the art (see for example U.S.application Ser. No. 12/064,014), A synthesis column was packed withsolid support derivatized with the first nucleoside residue (natural orchemically modified). Synthesis was initiated by detritylation of theacid labile 5′-O-dimethoxytrityl group to release the 5′-hydroxyl. Asuitably protected phosphoramidite and a suitable activator inacetonitrile were delivered simultaneously to the synthesis columnresulting in coupling of the amidite tip the 5′-hydroxyl. The column wasthen washed with a solvent, such as acetonitrile. An oxidizing solution,such as an iodine solution was pumped through the column to oxidize thephosphite triester linkage P(III) to its phosphotriester P(V) analog.Unreacted 5′-hydroxyl groups were capped using reagents such as aceticanhydride in the presence of 2,6-lutidine and N-methylimidazole. Theelongation cycle was resumed with the detritylation step for the nextphosphoramidite incorporation. This process was repeated until thedesired sequence was synthesized. The synthesis concluded with the final5′-terminus protecting group (trityl or 5′-O-dimethoxytrityl).

Upon completion of the synthesis, the solid-support and associatedoligonucleotide were dried under argon pressure or vacuum. Aqueous basewas added and the mixture was heated to effect cleavage of the succinyllinkage, removal of the cyanoethyl phosphate protecting group, anddeprotection of the exocyclic amine protection,

The following process was performed on single strands that do notcontain ribonucleotides. After treating the solid support with theaqueous base, the mixture was filtered to separate the solid supportfrom the deprotected crude synthesis material. The solid support wasthen rinsed with water, which is combined with the filtrate. Theresultant basic solution allows for retention of the5′-O-dimethoxytrityl group to remain on the 5′ terminal position(trityl-on).

For single strands that contain ribonucleotides, the following processwas performed. After treating the solid support with the aqueous base,the mixture was filtered to separate the solid support from thedeprotected crude synthesis material. The solid support was then rinsedwith dimethylsulfoxide (DMSO), which was combined with the filtrate.Fluoride reagent, such as triethylamine trihydrofluoride, was added tothe mixture, and the solution was heated. The reaction was quenched withsuitable buffer to provide a solution of crude single strand with the5′-O-dimetlioxytrityl group on the final 5′ terminal position.

The tritylon solution of each crude single strand was purified usingchromatographic purification, such as SPE RPC purification. Thehydrophobic nature of the trityl group permits stronger retention of thedesired full-length oligo than the non-tritylated truncated failuresequences. The failure sequences were selectively washed from the resinwith a suitable solvent, such as low percent acetonitrile. Retainedoligonucleotides were then detritylated on-column with trifluoroaceticacid to remove the acid-labile trityl group. Residual acid was washedfrom the column, a salt exchange was performed, and a final desalting ofthe material commenced. The full-length oligo was recovered in apurified form with an aqueous-organic solvent. The final product wasthen analyzed for purity (HPLC), identity (Maldi-TOF MS), and yield (UVA₂₆₀). The oligos were dried via lyophilization or vacuum condensation.

Annealing: Based on the analysis of the product, the dried oligos weredissolved in appropriate buffers followed by mixing equal molar amounts(calculated using the theoretical extinction coefficient) of the senseand antisense oligonucleotide strands. The solution was then analyzedfor purity of duplex by chromatographic methods and desired finalconcentration. If the analysis indicated an excess of either strand,then the additional non-excess strand was titrated until duplex* wascomplete. When analysis indicated that the target product purity hasbeen achieved the material was delivered and ready for use.

Further Synthesis Steps fear Commercial Preparations

Once analysis indicates that the target product purity has been achievedafter the annealing step, the material is transferred to the tangentialflow filtration (TFF) system for concentration and desalting, as opposedto doing this prior to the annealing step.

Ultrafiltration: The annealed product solution is concentrated using aTFF system containing an appropriate molecular weight cut-off membrane.Following concentration, the product solution is desalted viadiafiltration using Milli-Q water until the conductivity of the filtrateis that of water.

Lyophilization: The concentrated solution is transferred to a bottle,flash frozen and attached to a lyophilizer. The product is thenfreeze-dried to a powder. The bottle is removed from the lyophilizer andis now ready for use.

RT-gPCR Assays (Primary Screens and Dose-Response Curves)

Hepa1-6 cells were cultured in Dulbecco's Modified Eagle Mediumsupplemented with 10% fetal bovine serum, 1% penicillin-steptomycin, and1% sodium bicarbonate. These cells were plated in a 96-well cultureplates at a density of 3000 cells/well 24 hours prior to transfection.

Transfections were performed using Opti-MEM I Reduced Serum Media andLipofectamine RNAiMAX per the manufacturers' directions. Final siRNAconcentrations are 10 nM and 1 nM for primary screens. Final siRNAconcentrations for the dose-response curves (ssDRCs) range from 40 nM to0.002 nM along an 8-point, 4-fold titration curve.

Twenty-four hours post-transfection cells were washed withphosphate-buffered saline and processed using the TaqMan Gene ExpressionCells-to-CT™ Kit, per manufacturer's instructions, to extract RNA,synthesize cDNA, and perform RT-qPCR using an SSB or ApoB specificTaqman primer/probe set on an ABI Prism 7900HT Sequence Detector.

Reverse transcription conditions were as follows: 60 minutes at 37° C.followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2minutes at 50° C., 10 minutes at 9.5° C., followed by 40 cycles of 15seconds at 95° C. and 1 minute at 60° CGAPDH mRNA levels were used fordata normalization. Knockdown of SSB/ApoB was calculated as the two-foldchange in SSB/ApoB cDNA measured in experimentally-treated cellsrelative to the SSB/ApoB cDNA measured in non-targeting, control-treatedcells.

In vitro stability assays

20 μg/mL siNA was incubated with C57/BL6 mouse serum at 37° C. At 0, 2,and/or 4 hours an aliquot of serum/siNA sample was combined with anequal volume of lysis-loading buffer (a proprietary buffer supplied byvendor containing 2M urea. For siNAs with multiple phosphorothioates,lysis-loading buffer was supplemented with 10 mM DTT or 5 mM cysteineand TCEP to prevent oxygen replacement during the purification process)and mixed to quench the digestion. All quenched samples were incubatedon ice and an internal standard was added to control for variabilityduring the solid phase extraction (SPE) step.

SPE on lysis/siNA/serum samples was performed using 96-well PhenomenexSolid Phase Extraction plates containing 100 mg polymeric sorbent.Unless otherwise indicated, all SPE steps were performed using a vacuumsetting of ˜3″ Hg. The SPE wells were first conditioned with 1 mLmethanol, then equilibrated with 1 mL of equilibration buffer (50 mMNaH₂PO₄/2mM NaN₃, pH 5.5). Next, the lysis siNA/serum samples wereloaded onto the plate at a flow rate less than 1 mL/minute. After thesamples were loaded, the vacuum was increased to ˜10″ Hg briefly tocompletely evacuate the loading solution from the wells. The wells werethen washed six times with 1 mL of wash buffer (50 mMNaH₂PO₄/Acetonitrile, pH 5.5). Following the final wash the vacuum wasincreased to 15″ Hg for one minute to remove excess wash buffer. Next,samples were eluted into a sample collection tubes using 1 mL of washbuffer (100 mM NH₄HCO₄/10% THF/40% Acetonitrile, pH 8.8) per sample.After elution, samples were sealed with Airpore sealing tape and frozenon dry ice. Once frozen, samples were lyophilized overnight and storedat −20° C. until LC-MS analysis.

Lyophilized samples were reconstituted using 150 μL of 1 mM EDTA andbriefly vortexed. Reconstituted samples were then used to make 1:5dilutions in V-bottom MS 96-well plates using 1 mM. LC-MS analysis wasperformed using these 1:5 dilutions and a 10 μL injection volume.

LC/MS Analysis

Ion-pair reversed phase HPLC chromatographic separations were performedon a Thermo Hypersil Gold 30×2 mm C18 3 μ particle size column at a flowrate of 400 □L/min. Mobile phase A consisted of 1,7 mM TEA and 100 mMHEW (pH 7.5) in water, and mobile phase B was methanol:acetonitrile(90:10). A 10-20 □L injection of each sample was loaded onto the columnand separated using the following elution gradient: 5% B for one minute,5% to 25% B over two minutes, 90% B for one minute followed by initialconditions for two minutes for column re-equilibration.

Mass spectrometry was performed with a Thermo LTQ-Orbitrap XL orExactive Orbitrap system equipped with an electrospray ionization sourceand operating in the negative-ion mode, a MichromBioresources MSD4 HPLCpump, a Leap Technologies HTS PAL autosampler and a column heateroperated at 70″C. Data acquisition was performed with Thermo Excalibursoftware and data analysis was processed with in-house custom designedsoftware.

Calculations

The expression level of the gene of interest and % inhibition of geneexpression (% KD) was calculated using Comparative Ct method:

dCt=Ct _(Target) −Ct _(GAPDH)

ddCt(log 2fold change)=dCt _((Target SiNa)) dCt _((NTC))

Relative expression level=2^(−ddct)

% KD=100×(1−2^(−ddCt))

The non-targeting control siNA was, unless otherwise indicated, chosenas the value against which to calculate the percent inhibition(knockdown) of gene expression, because it is the most relevant control.

Additionally, only normalized data, which reflects the general health ofthe cell and quality of the RNA extraction, was examined. This was doneby looking at the level of two different mRNAs in the treated cells, thefirst being the target mRNA and the second being the normalized mRNA.This allowed for elimination of siNAs that might be potentially toxic tocells rather than solely knocking down the gene of interest. This wasdone by comparing the Ct for GAPDH in each well relative to the GAPDH Ctfor the entire plate.

In Vivo Knockdown Studies

C57/BL6 mice were dosed with siNA formulated in polymer conjugate orlipid nanoparticle at 3 mpk. Animals were sacked at the indicatedtimepoints and livers were harvested and stored at 4°C. in RNALateruntil ready for analysis.

Liver tissue was homogenized in Qiazol using stainless steel heads and aQiagen TissueLyser. Following homogenization, chloroform was added andsamples were centrifuged. The aqueous layer was combined with an equalvolume of 70% ethanol and samples were purified using a Qiagen RNeasypurification kit per manufacturer's directions. The resulting RNA wasthen normalized, cDNA was synthesized, and RT-qPCR was performed usingSSB or ApoB specific Taqinan primer/probe sets on an ABT Prism 7900HTSequence Detector.

Reverse transcription conditions were as follows: 60 minutes at 37° C.followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15seconds at 95° C. and 1 minute at 60° C. GAPHD mRNA levels were used fordata normalization. Knockdown of SSB/ApoB was calculated as the log2fold change in SSB/ApoB cDNA measured in experimentally-treated cellsrelative to the SSB/ApoB cDNA measured in non-targeting, control-treatedcells.

Synthesis of siNA Polyconjugates

General Polymer Synthesis—Continuous Cationic Polymerization

Continuous Synthesis of Vinyl Ether Polymers Using Three ReactingStreams

Stream 1: Octadecyl vinyl ether (6.31 g, 21.27 mmol, 1 eq), n-butylvinyl ether (8.52, 85.07 mmol, 4 eq) and N-(2-Vinyloxy-ethyl)phthalimide(69.30 g, 319.02. mmol, 15 eq) was dissolved in 900 mL ofdichloromethane (150 ml/g octadecyl vinyl ether) at a water contentbetween 50-100 ppm.

Stream 2: Boron trifluoride diethyl etherate (0.92 g, 6.48 mmol, 1.5 mol% vs monomers) was dissolved in 45 mL of dichloromethane.

Stream 3: 2M ammonia in methanol (12.96 mL, 25.91 mmol, 4 eq versusboron trifluoride diethyl etherate) was dissolved in 887 mL ofdichloromethane.

Stream 1 was pumped at 1.429 mL/min through 1/16″ PTFE and 316 stainlesssteel tubing introduced to a controlled bath set at −30° C. Stream 2 waspumped at 0.0714 mL/min through 1/16″ PTFE and 316 stainless steeltubing introduced to the controlled bath. Streams 1 and 2 are mixed in a1 mm ID 316 stainless steel tee before entering a 30 mL coil of ⅛″ 304stainless steel tubing. Stream 3 was pumped at 1.42.9 mL/min through1/16″ PTFE and 316 stainless steel tubing introduced to the controlledbath before mixing with the resulting stream from the 30 ML coil(mixture of Streams 1 and 2) in a 1 mm ID 316 stainless steel tee. Theresulting stream exited the controlled bath to a collection vessel. Thecollected polymer was isolated by removal of dichloromethane underreduced pressure to afford copolymer with a molecular weight of 29.4 kDa(add MW details) and polydispersity index of 2.1.

Polymer Deprotection, Purification and Characterization

In a 3-neck flask fitted with an overhead stirrer, reflux condenser, andnitrogen inlet was slurried Polymer 1 (50.0 g, 79 mmol) in 2-Propanol(1000 ml). Then charged with hydrazine (25% wt in H₂O) (499 ml, 3889mmol) and heated (65°C.). After 16 hrs, the reaction was cooled to roomtemperature. A constant volume distillation was performed to remove2-propanol while adding 0.1 M NaOH to maintain a volume of 1500 mL oftotal reaction volume. The distillation was continued until amount of2-propanol remaining in the reaction mixture was below 1 percent of thetotal volume as monitored by GC. The aqueous polymer solution was thensubjected to TFF purification (PALL centremate membrane, 1K MW cutoff,part number) with NaOH (0.25 N) until HPLC of solution indicatedcomplete removal of phthalhydrazide. Then used water until pH of wastestream became neutral (pH 7-8). The aqueous solution was thenfreeze-dried to obtain product (20.3 g) as a sticky oil. Water contentof the isolated polymer was determined by TGA. Sodium content of theisolated polymer was determined by ICP-MS. The weight percent of theisolated polymer was determined by subtracting the amount by weight ofwater and sodium hydroxide.

Synthesis of SATA-siNA First Conjugation Step

The siNA (1 g, 0.0714 mmol) was dissolved in 0.1M sodium bicarbonatebuffer (20 ml, 50 mg/mL) in a vial with magnetic stir bar and cooled to0-5° C. in an ice water bath. In a separate vial SATA (83 mg, 0.357mmol, 5 equivalents) was dissolved in 0.78 ml DMSO. The SATA solutionwas added over 1 min and the clear, colorless reaction mixture stirredat 0-5° C. for 2 h. After 2 h, the reaction mixture was sampled andanalyzed by IJPLC or HPLC for completion of the conjugation. If greaterthan 5% siNA remained unreacted, another charge of SATA in DMSO (2.0equivalents) was added and the reaction aged at 0-5° C. for completionof the SATA conjugation (confirmation by HPLC or UPLC). When there wasless than 5% unreacted siNA 1 remaining by UPLC or HPLC, the reactionmixture was purified by TFF (MW cutoff and manufacturer information)dialysis using endonuclease free water until HPLC indicated the removalof N-hydroxysuccinimide, and N-succinimidyl-S-acetylthioacetate. Therecovered solution was lyophilized to a white fluffy solid.

Activation Step: Activation of Polymer with SMPT

Polymer 1 (1.2 g) in a 40 mL vial was dissolved in 100 mM sterile TRISbuffer at pH 9 (120 Ml, 10 mg/mL) and added to a 1 L sterile plasticbottle. To this solution was added SMPT as 1 mg/mL solution in DMSO (18mg, 1800 uL) corresponding to L5 wt % with respect to the polymerweight. The solution was stirred for 1 hr at rt to generate activatedpolymer. The reaction was monitored for release of N-hydroxy succinimideby HPLC.

Second Conjugation Step—Conjugation of SATA-siNA to Activated Polymer

The activated polymer was further diluted using 100 mM sterile TRISbuffer at pH 9 (496 mL), followed by the addition of SATA-modified siNAas a solution in water (250 mg, 32.4 mg/mL, 7716 uL). This solution wasaged for 4 hours at room temperature. The reaction was monitored by HPLCfor release of 2-thiopyridine.

Masking Step: Formulation of siNA-Polymer Conjugate with Masking Agents

In a separate 1 L sterile plastic bottle, solti, CDM-NAG (5.5 g) andCDM-PEG (2.85 g) was added. The siNA-polymer conjugate solution wastransferred by pouring into the plastic bottle containing the CDM-NAGand CDM-PEG solids. The mixture was stirred for 2 minutes to dissolveall solids and then transferred by pouring into the original plasticbottle, which contained the siNA-polymer conjugate. The reaction wasstirred for 1 hour. The pH of the final solution was monitored to ensurethe pH was between 8-9. The reaction was monitored by SAX and SEC tovisualize the polymer conjugate, and determine the amount of siNAconjugated covalently to the polymer. The amount of CDM-NAG and. CDM-PEGwas determined by HPLC. The concentration of siNA in solution wasdetermined by ICP-MS, measuring phosphorous concentration.

Example 2 LNP Formulation of siNA General Process Description for LNPFormulations:

The lipid nanoparticles are prepared by an impinging jet process. Theparticles are formed by mixing lipids dissolved in alcohol with siNAdissolved in a citrate buffer. The mixing ratio of lipids to siNA istargeted at 45-55% lipid and 65-45% siNA. The lipid solution contains acationic lipid, a helper lipid (cholesterol), PEG (e.g. PEG-C-DMA,PEG-DMG) lipid, and DSPC at a concentration of 5-15 mg/mL with a targetof 9-12 mg/mL in an alcohol (for example ethanol). The ratio of thelipids has a mole percent range of 25-98 for the cationic lipid with atarget of 35-65, the helper lipid has a mole percent range from 0-75with a target of 30-50, the PEG lipid has a mole percent range from 1-15with a target of 1-6, and the DSPC has a mole percent range of 0-15 witha target of 0-12. The siNA solution contains one or more siNA sequencesat a concentration range from 0.3 to 1.0 mg/mL with a target of 0.3 -0.9mg/mL in a sodium citrate buffered salt solution with pH in the range of3.5-5. The two liquids are heated to a temperature in the range of15-40° C., targeting 30-40° C., and then mixed in an impinging jet mixerinstantly forming the LNP. The teeID has a range from 0.25 to 1.0 mm anda total flow rate from 10-600 mL/minute The combination of flow rate andtubing ID has the effect of controlling the particle size of the LNPsbetween 30 and 200 nm. The solution is then mixed with a bufferedsolution at a higher pH with a mixing ratio in the range of 1:1 to 1:3vol:vol but targeting 1:2 vol:vol. This buffered solution is at atemperature in the range of 15-40° C., targeting 30-40° C., The mixedLNPs are held from 30 minutes to 2 hrs prior to an anion exchangefiltration step. The temperature during incubating is in the range of15-40° C., targeting 30-40° C. After incubating, the solution arefiltered through a 0.8 um filter containing an anion exchange separationstep. This process uses tubing IDs ranging from 1 ram ID to 5 mm ID anda flow rate from 10 to 2000 mL/minute The LNPs are concentrated anddiafiltered via an ultrafiltration process where the alcohol is removedand the citrate buffer is exchanged for the final buffer solution suchas phosphate buffered saline. The ultrafiltration process uses atangential flow filtration format (TFF). This process uses a membranenominal molecular weight cutoff range from 30 -500 KD. The membraneformat is hollow fiber or flat sheet cassette. The TFF processes withthe proper molecular weight cutoff retains the LNP in the retentate andthe filtrate or permeate contains the alcohol; citrate buffer; and finalbuffer wastes. The TFF process is a multiple step process with aninitial concentration to a siNA concentration of 1-3 mg/mL. Followingconcentration, the LNPs solution is diafiltered against the final bufferfor 10-20 volumes to remove the alcohol and perform buffer exchange. Thematerial is then concentrated an additional 1-3 fold. The final steps ofthe LNP process are to sterile filter the concentrated LNP solution andvial the product.

Analytical Procedure:

1) siNA concentration

The siNA duplex concentrations are determined by Strong Anion-ExchangeHigh-Performance Liquid Chromatography (SAX-HPLC) using Waters 2695Alliance system (Water Corporation, Milford Mass. ) with a 2996 PDAdetector. The LNPs, otherwise referred to as RNAi Delivery Vehicles(LNPs), are treated with 0.5% Triton X-100 to free total siNA andanalyzed by SAX separation using a Dionex BioLC DNAPac PA 200 (4×250 mm)column with UV detection at 254 nm. Mobile phase is composed of A: 25 mMNaClO₄, 10 mM Tris, 20% EtOH, pH 7.0 and B: 250 mM NaClO₄, 10 mM Tris,20% EtOH, pH 7.0 with a liner gradient from 0-15 min and a flow rate of1 ml/minute. The siNA amount is determined by comparing to the siNAstandard curve.

2) Encapsulation Rate

Fluorescence reagent SYBR Gold is employed for RNA quantitation tomonitor the encapsulation rate of LNPs. LNPs with or without TritonX-100 are used to determine the free siNA and total siNA amount. Theassay is performed using a SpectraMax M5e microplate spectrophotometerfrom Molecular Devices (Sunnyvale, Calif. ). Samples are excited at 485nm and fluorescence emission is measured at 530 11M. The siNA amount isdetermined by comparing to an siNA standard curve.

Encapsulation rate=(1−free siNA/total siNA)×100%

3) Particle Size and Polydispersity

LNPs containing I pg siNA are diluted to a final volume of 3 ml with 1×PBS. The particle size and polydispersity of the samples is measured bya dynamic light scattering method using ZetaPALS instrument (BrookhavenInstruments Corporation, Holtsville, N.Y.). The scattered intensity ismeasured with He—Ne laser at 25° C. with a scattering angle of 90°.

4) Zeta Potential Analysis

LNPs containing 1 μg siNA are diluted to a final volume of 2 ml with 1mM Tris buffer (pH 7.4). Electrophoretic mobility of samples isdetermined using ZetaPALS instrument (Brookhaven InstrumentsCorporation, Holtsville, N.Y.) with electrode and He—Ne laser as a lightsource. The Smoluchowski limit is assumed in the calculation of zetapotentials.

5) Lipid Analysis

Individual lipid concentrations are determined by Reverse PhaseHigh-Performance Liquid Chromatography (RP-HPLC) using Waters 2695Alliance system (Water Corporation, Milford Mass.) with a Corona chargedaerosol detector (CAD) (ESA Biosciences, Inc, Chelmsford, Mass.).Individual lipids in LNPs are analyzed using an Agilent Zorbax SB-C18(50×4.6 mm, 1.8 μm particle size) column with CAD at 60° C. The mobilephase is composed of A: 0.1% TFA in H₂O and B: 0.1% TFA in IPA. Thegradient changes from 60% mobile phase A and 40% mobile phase B fromtime 0 to 40% mobile phase A and 60% mobile phase B at 1.00 min; 40%mobile phase A and 60% mobile phase B from 1.00 to 5.00 min; 40% mobilephase A and 60% mobile phase B from 5.00 min to 25% mobile phase A and75% mobile phase B at 10.00 min; 25% mobile phase A and 75% mobile phaseB from 10.00 min to 5% mobile phase ,A and 95% mobile phase B at 15.00min; and 5% mobile phase A and 95% mobile phase B from 15.00 to 60%mobile phase A and 40% mobile phase B at 20.00 min with a flow rate of 1ml minute. The individual lipid concentration is determined by comparingto the standard curve with all the lipid components in the LNPs with aquadratic curve fit. The molar percentage of each lipid is calculatedbased on its molecular weight.

General Formulation Procedure for CLinDMA/Cholesterol/PEG-DMG at a Ratioof 71.9:20.2:7.9.

Certain siNA solutions were prepared by dissolving siNAs in 25 mMcitrate buffer (pH 4.0) at a concentration of 0.8 mg/mL. Lipid solutionswere prepared by dissolving a mixture of 2S-Octyl-ClinDMA, cholesteroland PEG-DMG at a ratio of 71.9:20.2:7.9 in absolute ethanol at aconcentration of about 10 mg/mL. Equal volume of siNA and lipidsolutions were delivered with two syringe pumps at the same flow ratesto a mixing T connector. The resulting milky mixture was collected in asterile bottle. This mixture was then diluted slowly with an equalvolume of citrate buffer, and filtered through a size exclusion hollowfiber cartridge to remove any free siNA in the mixture. Ultra filtrationagainst citrate buffer (pH 4.0) was employed to remove ethanol (teststick from ALCO screen), and against PBS (pH 7.4) to exchange buffer.The final LNP was obtained by concentrating to a desired volume andsterile filtered through a 0.2 mm filter, The obtained LNPs werecharacterized in term of particle size, alcohol content, total lipidcontent, nucleic acid encapsulated, and total nucleic acidconcentration.

General LNP Preparation For Various Formulations in Table 18

siNA nanoparticle suspensions in Table 18 are prepared by dissolvingsiNAs and: or carrier molecules in 20 mM sodium citrate buffer (pH 5.0)at a concentration of about (0.40 mg/mL. Lipid solutions are prepared bydissolving a mixture of cationic lipid (e.g.,(13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, see structurein Table 19), DSPC, Cholesterol, and PEG-DIG (ratios shown in Table 18)in absolute ethanol at a concentration of about 8 mg/mL. The nitrogen tophosphate ratio was approximated to 6:1.

Nearly equal volumes of siNA/carrier and lipid solutions are deliveredwith two FPLC pumps at the same flow rates to a mixing T connector. Aback pressure valve wais used to adjust to the desired particle size.The resulting milky mixture is collected in a sterile glass bottle. Thismixture is then diluted with an equal volume of citrate buffer, followedby equal volume of PBS (pH 7.4), and filtered through an ion-exchangemembrane to remove any free siNA/carrier in the mixture. Ultrafiltration against PBS (7.4) ias employed to remove ethanol and toexchange buffer. The final LNP is obtained by concentrating to thedesired volume and sterile filtering through a 0.2 μm filter. Theobtained LNPs are characterized in terms of particle size, Zetapotential, alcohol content, total lipid content, nucleic acidencapsulated, and total nucleic acid concentration.

LNP Manufacture Process

In a non-limiting example, LNPs are prepared in bulk as follows. Theprocess consists of (1) preparing a lipid solution; (2) preparing ansiNA/carrier solution: (3) mixing/particle formation; (4) incubation;(5) dilution; (6) ultrafiltration and concentration.

1. Preparation of Lipid Solution

2L glass reagent bottles and measuring cylinders are depyrogenated. Thelipids are warmed to room temperature. Into the glass reagent bottle istransferred 8.0 g of(13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine with a pipetteand 1.2 g of DSPC, 3.5 g of Cholesterol, 0.9 g of PEG-DMG were added. Tothe mixture is added 1 L of ethanol. The reagent bottle is placed inheated water bath, at a temperature not exceeding 50° C. The lipidsuspension is stirred with a stir bar. A thermocouple probe is put intothe suspension through one neck of the round bottom flask with a sealedadapter. The suspension is heated at 30-40 C. until it became clear. Thesolution is allowed to cool to room temperature.

2. Preparation of siNA/Carrier Solution

Into a sterile container (Coming storage bottle) is weighed 0.4 g timesthe water correction factor (approximately 1.2) of siNA powder. The siNAis transferred to a depyrogenated 2 L glass reagent bottle. The weighingcontainer is rinsed 3× with citrate buffer (20 mM, pH 5.0) and therinses are placed into the 2 L glass bottle, QS with citrate buffer to 1L. The concentration of the siNA solution is determined with a UVspectrometer using the following procedure. 20 μL is removed from thesolution, diluted 50 times to 1000 μL, and the UV reading recorded atA260 nm after blanking with citrate buffer. This is repeated. Note, ifthe readings for the two samples are consistent, an average can be takenand the concentration calculated based on the extinction coefficients ofthe siNAs. If the final concentration is out of the range of 0.40±0.01mg/mL, the concentration can be adjusted by adding more siNA/carrierpowder, or adding more citrate buffer. This process can be repeated forthe second siNA, if applicable

When the siNA/carrier solution comprised a single siNA duplex instead ofa cocktail of two or more siNA duplexes and/or carriers, then thesiNA/carrier was dissolved in 20 mM citrate buffer (pH 5.0) to give afinal concentration of 0.4 mg/mL.

The lipid and ethanol solutions are then sterile filtered through a PallAcropak 20 0.8/0.2 μm sterile filter PN 12203 into a depyrogenated glassvessel using a Master Flex Peristaltic Pump Model 7520-40 to provide asterile starting material for the encapsulation process. The filtrationprocess is run at an 80 mL scale with a membrane area of 20 cm². Theflow rate was 280 mL/minute. This process can be scaled by increasingthe tubing diameter and the filtration area,

3. Particle Formation—Mixing Step

Using a two-barrel syringe driven pump (Harvard 33 Twin Syringe), thesterile lipid/ethanol solution and the sterile siNA/carrier orsiNA/carrier cocktail/citrate buffer (20 mM citrate buffer, pH 5.0)solutions are mixed in a 0.5mm ID T-mixer (Mixing Stage I) at equal, ornearly equal, flow rates. The resulting outlet LNP suspension contained40-50 vol % ethanol. To obtain a 45 vol % ethanol outlet suspension, thesterile lipid/ethanol and the sterile siNA/carrier or siNA/carriercocktail/citrate buffer solutions are mixed at flow rates of 54 mL/minand 66 mL/min, respectively, such that the total flow rate of the mixingoutlet is 120 mL/min

4. Dilution

The outlet stream of Mixing Stage I is fed directly into a 4 mm T-mixer(Mixing Stage II), where it is diluted with a buffered solution athigher pH (20 mM sodium citrate, 300 mM sodium chloride, pH 6.0) at aratio of 1:1 vol:vol %. This buffered solution is at a temperature inthe range of 30-40° C., and is delivered to the 4 mm T-mixer via aperistaltic pump (Cole Parmer MasterElex L/S 600 RPM) at a flow rate of120 mL/min

The outlet stream of Mixing Stage II is fed directly into a 6 mm IDT-mixer (Mixing Stage III), where it is diluted with a buffered solutionat higher pH (PBS, pH 7.4) at a ratio of 1:1 vol:vol%. This bufferedsolution is at a temperature in the range of 15-25° C., and is deliveredto the 6 mm T-mixer via peristaltic pump (Cole Partner MasterFlex L/S600 RPM) at a flow rate of 240 mL/min.

5. Incubation and Free siNA Removal

The outlet stream of Mixing Stage III is held after mixing for 30 minuteincubation. The incubation is conducted at temperature of 35-40° C. andthe in-process suspension is protected from light. Following incubation,free (un-encapsulated) siNA is removed via anion exchange with Mustang Qchromatography filters (capsules). Prior to use, the chromatographyfilters are pre-treated sequentially with flushes of 1N NaOH, 1M NaCl,and a final solution of 12.5 vol % ethanol in PBS. The p1-I of the finalflush is checked to ensure pH <8. The incubated LNP stream is thenfiltered via Mustang Q filters via peristaltic pump (Cole PartnerMasterFlex US 600 RPM) at flow rate of approximately 100 Ml/min. Thefiltered stream is received into a sterile glass container forultrafiltration and concentration as follows.

6. Ultrafiltration Concentration and Sterile Filtration

The ultrafiltration process is a timed process and the flow rates mustbe monitored carefully. This is a two step process; the first is aconcentration step taking the diluted material and concentratingapproximately 8-fold, to a concentration of approximately 0.3-0.6 mg/mLsiNA.

In the first step, a ring-stand with a ultrafiltration membrane 100 kDaPES (Spectrum Labs) installed is attached to a peristaltic pump(Spectrum KrosFloII System). 9.2 L of sterile distilled water is addedto the reservoir; 3 L is drained to waste and the remainder is drainedthrough permeate to waste. 5.3 L of 0.25 N sodium hydroxide is added tothe reservoir with 1.5 L drained to waste and 3.1 L drained throughpermeate to waste. The remaining sodium hydroxide is held in the systemfor sanitization (at least 10 minutes), and then the pump is drained.9.2 L of 70 (v/v)% isopropyl alcohol is added to the reservoir with L5 Ldrained to waste and the remainder drained through permeate to waste. 6L of conditioning buffer (12.5% ethanol in phosphate buffered saline) isadded with 1.5 L drained to waste and the remainder drained though thepermeate until the waste is of neutral pH (7-8). A membrane flux valueis recorded, and the pump was then drained.

The diluted LNP solution is placed into the reservoir to the 1.1 L mark.The pump is turned on at 2.3 L/min. After 5 minutes of recirculation,the permeate pump is turned on at 62.5 ML/min and the liquid level isconstant at approximately 950 mL in the reservoir. The diluted LNPsolution is concentrated from 9.8 L to 1.1 L in 140 minutes, and thepump is paused when all the diluted LNP solution has been transferred tothe reservoir.

The second step is a diafiltration step exchanging the ethanol/aqueousbuffer to phosphate buffered saline. During this step, approximately10-20 diafiltration volumes of phosphate buffered saline are used.Following diafiltration, a second concentration is undertaken toconcentrate the LNP suspension 3-fold to approximately 1-1.5 mg/mLsiRNA. The concentrated suspension is collected into sterile, plasticPETG bottles. The final suspension is then filtered sequentially viaPall 0.45 um PES and Pall 0.2 um PES filters for terminal sterilizationprior to vial filling.

The obtained LNPs are characterized in terms of particle size, Zetapotential, alcohol content, total lipid content, nucleic acidencapsulated, and total nucleic acid concentration.

Synthesis of Novel Cationic Lipids

Synthesis of novel cationic lipids of the invention is a linear processstarting from lipid acid (i). Coupling to N,O-dimethyl hydroxylaminegives the Weinreb amide ii. Grignard addition generates ketone iii.Titanium mediated reductive amination gives final products of type iv.

Synthesis of the single carbon homologated cationic lipids v is a linearprocess starting from lipid ketone (iii). Conversion of the ketone tothe nitrile (iv) is accomplished via treatment with TOSMIC and potassiumtern-butoxide. Reduction of the nitrile to the primary amine followed byreductive amination provides final cationic lipids v.

Synthesis of two carbon homologated cationic lipids viii is a linearprocess starting from lipid ketone (iii). Conversion of the ketone tothe α,β-unsaturated amide vi is accomplished under Peterson conditions.Conjugate reduction of the α,β-unsaturation is performed usingLS-Selectride to give amide vii. Reduction of the amide with lithiumaluminum hydride provides final cationic lipids viii.

Cyclopropyl containing lipids are prepared according to General Scheme4. Unsaturated Weinreb amides ii are subjected to Simmons-Smithcyclopropanation conditions to give cyclopropyl containing Weinrebamides ix. These are carried on to final products as outlined in GeneralSchemes 1-3.

Synthesis of allylic amine cationic lipids xv is a linear processstarting with aldehyde x. Addition of t-butyl aceate generates β-hydroxyester xi. Conversion of the hydroxyl functionality to a fluoro groupfollowed by acid treatment generates β-fluoro acid xii. Conversion ofthe acid to the Weinreb amide followed by Grignard addition gives theβ-fluoro ketone xiv. Reductive amination results in simultaneouselimination to generate the desired allylic amine xv.

20,23-nonacosadien-10-amine, N,N-dimethyl-, (20Z,23Z) (Compound 1)

11 ,14-Eicosadienoic acid, (11Z,14Z)-(50 g, 16:2 mmol),N,O-Dimethylhydroxylamine hydrochloride (31.6 g, 324 mmol), HOAt (44.1g, 324 mmol), Et₃N (45.2 mL, 324 mmol), and EDC (62.1 g, 324 mmol) weremixed in DCM (810 mL) and stirred overnight at ambient temperature.Reaction was then washed 5×700 mL water, then washed 1×600 mL 1 M NaOH,dried with sodium sulfate, filtered through celite and evaporated toobtain 53.06 g (93%) 11,14-eicosadienamide, N-methoxy-N-methyl-,(11Z,14Z) as a clear golden oil. ¹H NMR (400 MHz, CDCl₃) δ 5.35 (m, 4H),3.68 (s, 3H), 3.18 (s, 3H), 2.77 (m, 2H), 2.41 (t, J=7 Hz, 2H), 2.05 (m,4H), 1.63 (m, 2H), 1.40-1.26 (m, 18H), 0.89 (t, J=7 Hz, 3H).

11,14-eicosadienamide, N-methoxy-N-methyl-, (11Z,14Z)-1 (4 g, 11.38mmol) was dissolved in dry THF (50.0 ml) in a 250 mL flask then 1 Mnonylmagnesium bromide (22.76 ml, 22.76 mmol) was added under nitrogenat ambient temperature. After 10 min, the reaction was slowly quenchedwith excess sat. aq NH₄Cl. The reaction was washed into a separatoryfunnel with hexane and water, shaken, the lower aqueous layer discarded,the upper layer dried with sodium sulfate, filtered, and evaporated togive crude ketone as a golden oil. To the above crude ketone was addeddimethylamine (2 M in THF) (14.22 ml, 28.4 mmol) followed by Ti(O-i-Pr)₄(6.67 ml, 22.76 mmol) and let stir overnight. The next day, added EtOH(50 ml) followed by NaBH₄ (0.646 g, 17.07 mmol). After 5 min ofstirring, directly injected entire reaction onto a 40 g silica columnthat was in line with a 330 g silica column. Eluted 10 min 100% DCM,then 30 min 0-15% MeOH/DCM, collected 20,23-nonacosadien-10-amine,N,N-dimethyl-, (20Z,23Z) (1) (2.45 g, 5.47 mmol, 48.1% yield) as afaintly golden oil. ¹H NMR (400 MHz, CDC1₃) δ 5.35 (m, 4H), 2.78 (m,2H), 2.23 (m, 1H), 2.21 (s, 6H), 2.05 (m, 4H), 1.45-1.16 (m, 38H), 0.89(m, 6H). HRMS calcd for C31H61N 448.4877, found 448.4872.

Compounds 2-30 are novel cationic lipids and were prepared according tothe. General Scheme 1 above.

Compound Structure HRMS 2

calcd C28H56N 406.4407, found 406.4405. 3

calcd C27H54N 392.4251, found 392.4250. 4

calcd C24H48N 350.3781, found 350.3770. 5

calcd C23H46N 336.3625, found 336.3613. 6

calcd C25H50N 364.3938, found 364.3941. 7

calcd C26H52N 378.4094, found 378.4081. 8

calcd C29H58N 420.4564, found 420.4562. 9

calcd C26H52N 378.4094, found 378.4089. 10

calcd C25H50N 364.3938, found 364.3931. 11

calcd C30H60N 434.4720, found 434.4717. 12

calcd C29H58N 420.4564, found 420.4561. 13

calcd C28H56N 406.4407, found 406.4404. 14

calcd C27H54N 392.4251, found 392.4245. 15

calcd C33H66N 476.5190, found 476.5196. 16

calcd C32H64N 462.5033, found 462.5045. 17

caled C29H59N 422.4720, found 422.4726. 18

calcd C28H57N 408.4564, found 408.4570. 19

calcd C30H59N 434.4720, found 434.4729. 20

calcd C29H61N 424.4877, found 424.4875. 21

calcd C32H64N 462.5033, found 462.5023. 22

calcd C33H64N 474.5033, found 474.5033. 23

calcd C29H60N 422.4720, found 422.4716. 24

calcd C29H60N 422.4720, found 422.4718. 25

calcd C31H64N 450.5033, found 450.5031. 26

calcd C31H64N 450.5033, found 450.5034. 27

calcd C35H72N 506.5659, found 506.5635. 28

calcd C31H64N 450.5033, found 450.5037. 29

calcd C33H68N 478.5346, found 478.5358. 30

calcd C27H56N 394.4407, found 394.4407.

(12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine (Compound 31)

A solution of keton iii (4.0 g, 9.55 mmol), TOSMIC (2.4 g, 12.4 mmol) indimethoxyethane (45 mL) was cooled to 0° C. and treated with potassiumCert-butoxide (19.1 mmol, 19.1 mL of a 1M solution in tBuOH). After 90minutes, the reaction was partitioned between hexanes and water. Theorganics were washed with water, dried over sodium sulfate, filtered andevaporated in cacao. This material was purified by flash chromatography(0-5% EtOAc/hexanes) to give desired product (containing ˜20% of s.m.).This mixture was carried into next step as is. LC/MS (M+H)=430.6.

Lithium aluminum hydride (23.9 mmol, 23.9 mL of a 1M solution in THF)was added directly to nitrile iv (3.42 g, 8 mmol) at ambient temperatureand the reaction was stirred for 20 minutes. The reaction was dilutedwith 100 mL THF, cooled to 0° C. and carefully quenched with sodiumsulfate decahydrate solution. The solids were filtered off and washedwith THF. The filtrate was evaporated in vacuo and carried directly intonext reaction crude. LC/MS (M+H)=434.6.

A solution of primary amine (3.45 g, 6.2mmol) in dichloroethane (100 mL)was treated with formaldehyde (1.6 mL, 21.7 mmol) followed by sodiumtriacetoxyborohydride (6.6 g, 31 mmol). After 5 minutes, the reactionwas partitioned between dichloromethane and 1N NaOH. The organics weredried over sodium sulfate, filtered and evaporated in vacua. The crudemixture was purified b reverse phase preparative chromatography (C8column) to provide (12Z, 15Z)-N,N-dimethyl-2-nonylhenicosa-12 ,15-dien-1-amine, HRMS calc'd 462.5033, found 462.5026. ¹H NMR (400 MHz, CDCl₃) δ5.35 (m, 4H), 2.78 (2H, t, J=5.6Hz), 2.18 (s, 6H), 2.05 (m, 6H), 1.3 (m,39H), 0.89 (m, 6H).

(13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (Compound 32)

The silyl amide Peterson reagent (3.1 g. 16.7 mmol) was dissolved in THE(35 mL.) and cooled to −63° C., To this solution was added nBuLi (1.6.7mmol, 6.7 mL of a 2.5M solution). The reaction was warmed to ambienttemperature for 30 minutes. The ketone (5.0 g, 11.9 mmol) was dissolvedin THF (25mL) in a second flask. The Peterson reagent was transferred tothe ketone solution at −60° C. The reaction was warmed to −40° C. for 1hour, then warmed to 0° C. for 30 minutes. The reaction was quenchedwith sodium bicarbonate, diluted with additional water and partitionedbetween water/hexanes. The organics were washed with brine, dried oversodium sulfate, filtered and evaporated in vacuo. Purification by flashchromatography (0-40% MTBE/hexanes) gave α,β-unsatured amide vi. ¹H NMR(400 MHz, CDCl₃) δ 5.75 (s, 1H), 5.36 (m, 4H), 3.01 (s, 3H), 2.99 (s,3H), 2.78 (t, 2H), 2.28 (t, 2H), 2.05 (m, 6H), 1.35 (m, 34H), 0.89 (m,6H).

α,β-unsatured amide vi (1 g, 2.1 mmol) and LS-Selectride (4.1 mmol, 4.1mL of a 1M solution) were combined in a sealed tube and heated to 60°C.for 24 hours. The reaction was cooled to ambient temperature andpartitioned between ammonium chloride solution and heptane. The organicswere dried over sodium sulfate, filtered and evaporated in maw to giveamide vii. This intermediate was carried directly into next reactioncrude.

To a solution of amide vii (2.85 g, 5.8 mmol) was added lithium aluminumhydride (8.7 mmol, 8.7 mL of a 1M solution). The reaction was stirred atambient temperature for 10 minutes then quenched by slow addition ofsodium sulfate decahydrate solution. The solids were filtered and washedwith THF and the filtrate evaporated in vacuo. The crude mixture waspurified by reverse phase preparative chromatography (C8 column) toprovide (13Z, 16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine(Compound 32) as an oil. HRMS (M+H) calc'd 476.5190, found 476.5189. ¹HNMR (400 MHz, CDCl₃) δ 5.37 (m, 4H), 2.78 (t, 2H), 2.42 (m, 8H), 2.05(q, 4H), 1.28 (m, 41H), 0.89 (m, 6H).

N,N-dimethyl-1-(2-octylcyclopropyl)heptadecan-8-amine (Compound 33)

To a solution of oleic acid (1 g, 3.5 mmol) in DCM (500 mL) cooled to 0°C. was added CDI (0.63 g, 3.9 mmol). The reaction was warmed to ambienttemperature for 30 minutes before cooling to 0° C. and treating firstwith triethylamine (0.39 g, 3.9 mmol) and then dimethyl hydroxylaminehydrochloride (0.38 g, 3.9 mmol). After 1 hour the reaction waspartitioned between water and heptane. The organics were dried overmagnesium sulfate, filtered and evaporate in vacuo to give crude Weinrebamide ii which was carried directly into next reaction.

A solution of diethylzinc (70.3 mmol, 70.3mL of a 1M solution) indichloromethane (130 mL) was cooled to −1°C. and treated dropwise withTFA (8.0 g, 70.3 mmol). After 30 minutes, diiodomethane (18.8 g, 70.3mmol) was added and this was aged for 30 minutes in the ice bath. Tothis solution was added Weinreb amide ii (7.6 g, 23.4 mmol). Thereaction was warmed to ambient temperature and stirred for 1 hour. Thereaction was quenched with ammonium chloride solution (100 mL) andorganic layer partitioned off, washed with 10% sodium thiosulfate, driedover magnesium sulfate, filtered and evaporated in vacua. Purificationwas flash chromatography (0-30% MTBE/heptane) gave desired product ix.¹H NMR (400 MHz, CDCl₃) δ 3.72 (s, 3H), 3.22 (s, 3H), 2.48 (t, 2H), 1.65(m, 2H), 1.39 (m, 22H), 1.18 (m, 2H), 0.91 (t, 3H), 0.68 (m, 2H), 0.59(m, 1H), −0.32 (m, 1H).

Conversion of Weinreb amide ix to Compound 33 was carried out in amanner analogous to that described for Compound 1 above (nonyl Grianardaddition followed by reductive amination). LC/MS (M+H)=436.6. ¹H NMR(400 MHz, CDCl₃) δ 2.25 (s, 6H), 1.30 (m, 45H), 0.91 (m, 6H), 0.68 (m,2H), 0.59 (m, 1H), −0.31 (m, 1H).

Compounds 34-43 are novel cationic lipids and were prepared according toGeneral Schemes 1-4 above.

Compound Structure HRMS 34

calcd C30H62N 436.4877, found 436.4872. 35

calcd C32H66N 464.5190, found 464.5186. 36

calcd C34H70N 492.5503, found 492.5496. 37

calcd C33H66N 476.3190, found 476.5174. 38

calcd C29H60N 422.4720, found 422.4701. 39

calcd C30H62N 436.4877, found 436.4880. 40

calcd C32H66N 464.5190, found 464.5199. 41

calcd C30H62N 436.4877, found 436.4877. 42

calcd C30H62N 436.4877, found 436.4875. 43

LC/MS (M + H) 408.6.

(11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,23-trien-10-amine (Compound 44)

To a solution of LDA (95 mmol, 47.5 mL of a 2M solution) in THF (127 mL)cooled to −78° C. was added t-butyl acetate. The reaction was stirredfor 15 minutes followed by addition of aldehyde x. The reaction wasimmediately quenched with ammonium chloride solution, warmed to ambienttemperature and partitioned between water/pentane. The organics weredried over sodium sulfate, filtered and evaporated in vacuo. LEMS(M+H−tBu)=325.4.

Hydroxy ketone xi (7 g, 18.4 mmol) was dissolved in dichloromethane (i50mL) and cooled to 0° C. and treated with deoxofluor (7.3 g, 33.1 mmol).The reaction was warmed to ambient temperature with stirring for 16hours followed by quenching with sodium bicarbonate solution. Thereaction was partitioned and the organics dried over sodium sulfate,filtered and evaporate in vacuo. Flash column chromotagraphy ((h5% ethylacetate/hexanes) gave the □-fluoro ester.

Fluoro ester intermediate (6 g, 15.6 mmol) in dichloromethane wastreated with hydrogen chloride (157 mmol, 39.2 mL of a 4M solution indioxane) and the reaction was stirred at ambient temperature for 16hours. The reaction was evaporated in vacuo to give desired β-fluoroacid xii. LC/MS (M+H)=327.3.

Fluoro carboxylic acid xii (5.1 g, 15.7 mmol), EDC (6.0 g, 31.4 mmol)),N,O-dimethylhydroxylamine hydrochloride (3.1 g, 31.4 mmol.),trimethylamine (4.0 g, 39.2 mmol), and HOAt (4.3 g, 31.4 mmol)) werecombined in DCM (78 mL) and stirred at ambient temperature for 16 hours.The reaction was partitioned between water/DCM and the organics werewashed with water (3×) and NaOH solution (1×), dried over sodiumsulfate, filtered and evaporated in vacua. Crude material was purifiedby reverse phase preparative chromatography to give desired Weinrebamide xiii. LC/MS(M+H)=370.4.

A solution of Weinreb amide xiii (4.3 g, 11.7 mmol) in THF (50 mL) wastreated with nonylmagnesium bromide (23.4 mmol, 23.4 mL of a 1Msolution) at ambient temperature. The reaction was quenched withammonium chloride solution after 1 hour and partitioned between waterand pentane. The organics were dried over sodium sulfate, filtered andevaporated in vacuo. This material was carried into next step crude.

Ketone xiv (5.1 g, 11.7 mmol) was treated with dimethylaraine (29.3mmol, 14.7 mL of a 2M solution in THF) and titanium(W) isopropoxide (6.7g, 23.5 mmol)) and the reaction was stirred at ambient temperature for16 hours. To the reaction mixture was added ethanol (50 mL) followed bysodium borohydride (0.67 g, 17.6 mmol)). The reaction was loadeddirectly onto a silica column and purified by flash chromatography(0-15% MeOH/DCM). The material required a second purification bypreparative reverse phase chromatography to give(11E,20Z,23Z)-N,N-dimethylnonacosa- 11,20,23- trien-10-amine. HRMScalc'd 446.4720, found 446.4724. ¹H NMR (400 MHz, CDCl₃) δ 5.48 (m, 1H),5.37 (m, 4H), 5.23 (m, 1H), 2.78 (t, 2H). 2.58 (m, 1H), 2.22 (s, 6H),2.04 (m, 6H), 1.56 (m, 1H), 1,30 (m, 31H), 0.89 (m, 6H).

Compound 45 is DLinKC2DMA as described in Nature Biotechnology, 2010,28, 172-176, WO 2010/042877 A1, WO 2010/048536 A2, WO 2010/088537 A2,and WO 2009/127060 A1.

Compound 46 is MC3 as described in WO 2010/054401, and WO 2(110/144740A1.

E. Lipid Nanoparticle Compositions

The following lipid nanoparticle compositions (LNPs) of the instantinvention are useful for the delivery of oligonucleotides, specificallysiNA molecules of the invention:

-   Cationic Lipid Cholesterol/PEG-DMG 56.6/38/5.4;-   Cationic Lipid/Cholesterol/PEG-DMG 60/38/2:-   Cationic Lipid/Cholesterol/PEG-DMG 67.3/29/3.7;-   Cationic Lipid/Cholesterol/PEG-DMG 49.3/47/3.7;-   Cationic Lipid/Cholesterol/PEG-DMG 50.3/44.3/5.4;-   Cationic Lipid/Cholesterol PEG-C-DMA/DSPC 40/48/2/10;-   Cationic Lipid/Cholesterol/PEG-DMG/DSPC 40/48/2/10; and-   Cationic Lipid/Cholesterol/PEG-DMG/DSPC 58/30/2/10.

Example 3 Abrogation of Immunogenicity with 2′-Sugar Modifications

Unmodified RNAs, including siRNAs, induce a immunostimulatory responsewhich is primarily mediated by endosomal Toll-like receptors (see Judgeand MacLachlan, 2008, Hum Gene Ther 19, 111-24.). The subsequent immuneresponse and release of inflammatory cytokines represents one challengefor the development of safe RNAi therapeutics. Previously publishedreports have investigated the immunostimulatory potentials of modifiedsiRNAs. Judge et al evaluated 2′OMe modification of all four nucleotidesin the passenger strand of an ApoB siRNA (see Judge et al., 2006, MolTher 13, 494-505). They report that A, G, and U modifications areeffective at reducing TNF-alpha levels however 2′OMe C was surprisinglyineffective. A subset of modified siRNAs were tested in vivo in mice andconfirmed the in vitro PBMC result, namely 2′OMe modifications areeffective unless applied to cytidine. The cytidine result wasrecapitulated in a separate study which also showed thatinterferon-alpha induction by an unmodified RNA can be antagonized by ansiRNA containing 2′OMe adenosines (see Eberle, et al., 2008, J Immunol180, 3:229-37).

Previously, 2′-Methoxy (2′OMe), fluoro (2′F), and deoxy (2′H) modifieduridines were compared in single strand RNAs and all three modificationtypes reduced TNA-alpha levels (see Sioud et al., 2007, Biochem BiophysRes Commun 361, 122-6). Interestingly only the 2′OMe modificationsignificantly antagonized the INFa induction by a separate unmodifiedRNA. Robbins et al., conducted a related experiment, evaluating theeffectiveness of 2′OMe modified A, G, and C in single strand RNAs (seeRobbins et al., 2007, Mol Ther 15, 1663-9). All three 2′OMemodifications effectively silenced the IFNa. induction of the RNAsthemselves however only 2′OMe-A completely antagonized IFNa induction bya separate unmodified RNA.

Duplex siRNAs containing combinations of 2′ F and 2′OMe modificationswere compared in vivo in mice (see Shin et al., 2007, Biochem BiophysRes Commun 364, 436-42). Overall methoxy modifications alone or combinedwith fluoro pyrimidines were effective at quieting interferon inductionwhile fluoro-only pyrimidine modifications were less effective. Cekaiteet al., employed a mRNA biomarker approach to evaluate the effects of2?- IJ and 2′ONTe-U modifications on the immunostimulatory potential ofsingle strand RNAs, finding that fluoro and methoxy uridine were equallyeffective at silencing the immune response (see Cekaite et al., 2007, JMol Biol 365, 90-108), In summary the body of published literature hasevaluated 2′OMe and 2? modifications in a variety of contexts but hasnot yet systematically compared 2? and 2′OMe modifications on all fournucleotides.

Here, applicant reports on the application of an in vitro humanperipheral blood monocyte (PBMC) assay to measure TNF-alpha inductionresulting from the administration of lipid nanoparticle (LNP) formulatedsiRNAs (see for example, Peacock et al., 2011, J Am Chem Soc 133,9200-3). In a systematic screen, 2′OMe and 2′F modifications wereapplied in a nucleotide specific manner to either guide, passenger, orboth strands of the duplex of multiple siRNAs and assayed for immunestimulation. Applicant adds to existing reports that nucleotide biasesinfluence the ability of these ribose modifications to confer immunestealth by recapitulating known liabilities of modifying cytidine anddiscovers that adenosine was the only nucleotide to confer immunestealth by both 2′OMe and 2′F ribose modifications.

MATERIALS AND METHODS

Oligo sequence and synthesis: Beta-galactosidase siRNAs based onpreviously published siRNA sequences (see Judge et al., 2005, NatBiotechnol. 23, 457-62) with the addition of two nucleotide uridineoverhangs on both strands (R-008242441-000D, see Table 1). The originalpublished siRNAs were blunt without overhangs. The B-gal control siRNAused is a non-targeting control sequence (R-008384290-000L). A morelimited analysis was conducted with a previously published siRNAtargeting ApoB (see Judge et al., 2006, Mol Ther 13, 494-505) having aphosphorylated guide strand (R-008384421-000T). Modified siRNAs weresynthesized at Merck & Co. using standard methods.

siRNA formulation and administration to PBMCs: Isolation of humanperipheral blood monocytes (PBMC), formulation of siRNA lipid nanoparticles (LNP), and administration to PBMCs was performed as previouslydescribed (see Peacock et al., 2011, J Am Chem Soc 133, 9200-3). PBMCswere purified from buffy coats (leukocytes & platelets) over aFicoll-Paque gradient (Amersham). Cells were resuspended in freezingmedia (Gibco) at a concentration of 5-10 million cells per ml, aliquotedto cryogenic vials (Corning) and frozen overnight at −70C beforetransferring to appropriate liquid nitrogen cell storage system. L201lipid mixture is a combination of 6% Peg-DMG (Sunbright), 44%Cholesterol (MP Biomedical), and 50% ClinDMA (see U.S. Pat. No.7,514,099). Lipid mixture was sonicated until complete dissolution (˜5minutes) then cooled to room temperature. Volume was adjusted to 25 mLwith ethanol then transferred to a 50 mL conical tube and stored at 4 Cfor up to 1 week. siRNAs were diluted in HEPES Buffered Saline (20 mMHepes, 150 mM NaCl) then mixed at 3000 RPM in a plate shaker. Whileshaking, an equal volume of 200 uM stock siRNA was combined with L201lipid mixture in Costar 96-well round bottom plates. Samples werediluted with equal volume DPBS, mixed on low setting (700 RPM) for atleast 5 minutes. This generates LNP formulated siRNA at 50 uM. FrozenPBMC cells were thawed, diluted to desired concentration of 250 to 500thousand per well, and then cultured in RPMI media (Cellgro) containing2X PenStrep and 10% PBS. LNP-siRNA mixture was diluted 20-fold intoPBMCs freshly plated in Costar 96-well round bottom plates. Wells werequickly mixed with gentle pipetting then incubated overnight (16-20hrs).The next day, cells were pelleted and conditioned media was aliquotedfrom the experiment plate to a fresh 96-well storage plate. Samples werethen frozen at −80 or processed directly with cytokine ELISAs.

Cytokine ELISA assays: A white PS 96-well microplate (Nunc MaxiSorp) wascoated with 100 uL/well of human TNF-alpha antibody (Thermo Scientific)diluted 1:250 in PBS. Plate was sealed and incubated overnight on thebenchtop at room temperature. Plate was then blocked with 225 uL/well 4%BSA-PBS (blocking buffer) for 1 hr, RT. After overnight incubation,blocking buffer was aspirated from plates. Human TNFa standard wsaprepared from 2000 pg/mL stock as a 7-point, 2-fold serial dilution in10% PBS-PBS into the coated plate. 80-100 uL/well of PBMC supernatantwas transferred to the coated plate. Plate was incubated for 1-2 hr atroom temp with gentle shaking. Plate was washed 3× with PBST then 100uL/well of Detection Antibody diluted 1:250 in 4% BSA-PBS was added.Plate was sealed and incubated 2hr at RT (can also incubate ON at 4 C).Plate was washed 3× with PBST then 100 uL/well of strepavidin-HRPconjugate diluted 1:500 in 4% BSA-PBS was added and incubated for atleast 30 min. Plates were then washed 4× with PBST. Luminol-basedhorseradish peroxidase detection reagent, SuperSignal West PicoChemiluminescent Substrate (Pierce) was prepared by mixing equal partsLuminol/Enhancer in the Stable Peroxide Buffer. 100 uL ECL reagent wasadded to each well, mixed gently for 30 sec-1 min and then plate wasread on the EnVision plate reader (Perkin Elmer).

Measurement of Beta-galactosidase activity: A mouse hepatocyte derivedcell. line (Hepa1-6) was co-transfected with a Beta-galactosidaseplasmid (pCMV SPORT Beta-gal, Invitrogen) and siRNAs using Lipofectamine2000 (Invitrogen). Cells were seeded at 20,000 per well in 96-wellPolySorp opaque white plates (Nunc), incubated 24 hours at 37C, thentransfected with 10 nM siRNA and 0.6 ng/ul of pCMV SPORT Beta-galplasraid. Transfected cells were incubated overnight at 37C thenBeta-gal enzyme activity was measured using the Gal-Screen luminescencedetection system (Applied Biosystems) and. SpectraMax plate reader(Molecular Devices). Luminescence values were normalized to anon-targeting control siRNA to calculate percent activity. The controlsiRNA sequence contains fluoro 2′F (f), methoxy 2′OMe (m), deoxy 2′H (d)and ribo 2′OH (r) residues at the indicated positions as well asinverted abasic caps (iB) on the passenger strand (R-008039829-001W).

RESULTS

An in vitro assay was developed to measure the immunostimulatorypotential of siRNAs by monitoring the induction of tumor necrosis factoralpha (TNFa) in human peripheral blood monocytes (PBMC) (see Peacock etal., 2011, J Am Chem Soc 133, 9200-3). The siRNAs were formulated inlipid nanoparticles that approximate those used for in vivo studies andthe development of therapeutic siRNAs (see Abrams et al., 2010, Mol.Ther., 18: 171-80). Two published siRNAs with known immunostimulatorypotentials (see Judge et al., 2005, Nat Biotechnol 23, 457-62) wereselected for systematic evaluation of methoxy (2′OMe) and fluoro (2′F)ribose modifications of the four nucleotides of the passenger and guidestrands. One of these siRNAs targets Beta-galactosidase (B-gal 728)while the other is a related but non-targeting control (B-gal control).Additionally applicant conducted a more limited 2′OMe analysis of anApoB siRNA, another siRNA with previously described immune response (seeJudge et al., 2006, Mol. Ther., 13: 494-505).

Evaluation of pyrimidine modifications: Methoxy modification (2′OMe) ofcytidine was largely ineffective in reducing siRNA mediated immunestimulation as measured by elevation of TNFa levels in human PBMCcultures (Tables 14-17). This recapitulates published reports findingthat 2′OMe modification of cytidine was not effective (see .Eberle etal., 2008, J Immunol 180, 3229-37 and Shin et al., 2007, Biochem BiophysRes Commun 364, 436-42). We extend these observations to 2′-fluorocytidine modifications (2′F) which are equally ineffective in quietingsiRNA mediated TNFa induction. Uridine modification with 2′OMe ismarkedly more effective at reducing siRNA mediated immune stimulation(Tables 14-16); reducing TNFa levels 7-70 fold relative to unmodifiedcontrol (Table 14). However, 2′F modifications resulted in significantlyless reduction of TNFa levels (4-6 fold). Also of note, when the guidestrand of the B-gal control siRNA was modified at only three positions,2′F and 2′OMe conferred immune stealth is significantly compromised(Table 15). Methoxy modifications of pyrimidines in an ApoB siRNAresulted in similar trends: modified uridine reduced TNFa inductionwhile cytidine did not (Table 17).

Evaluation of purine modifications: Modification of guanosine followed asimilar pattern to that seen for uridine. Methoxy modifications areeffective at reducing TNFa induction, with as few as two 2′0111e Gresidues on the passenger or guide strand reducing immune stimulation˜15-fold (Tables 14-18). As with uridine, 2′F modifications were noteffective at significantly reducing TNFa levels on a consistent basis(Table 14) though modestly lower levels of TNFa were observed for theB-gal control siRNA (Table 15). Strikingly, either methoxy or fluoromodifications of adenosine significantly reduced TNFa levels in PBMCs(Tables 14-17). As few as three modified adenosines on the passengerstrand of B-gal control siRNA could reduce TNFa levels 7-fold (2′F) or47-fold (2′OMe) (Table 14). Methoxy modified purines were also effectiveat reducing TNFa induction for an ApoB siRNA (Table 17). Overall 2′OMemodified adenosines reduced TNFa levels ˜50-fold while 2′F modificationsreduced levels 4 to 13-fold. While 2′F mediated reduction of TNFa levelswas significantly less pronounced than corresponding 2′OMemodifications, 2′F adenosine modifications were far more effective thanguanosine, uridine, or cytidine.

Effect of 2′ modifications on siRNA knockdown: Knockdown activity forsiRNAs was measured using an in vitro cell-based assay relying onco-transfection of a plasmid containing a Beta-galactosidase transgenetogether with individual siRNAs. Beta-galactosidase enzymatic activitywas measured with a luminescence based detection system and normalizedto a non-targeting control siRNA (see Methods). The unmodified B-gal 728siRNA reduced enzyme activity levels to 18% relative to control. Theactivity of methoxy and fluoro modified versions of the 728 siRNA werecompared (Table 16). Generally 2′F modifications were broadly toleratedand did not negatively impact the potential of knockdown for this siRNA.However, 2′OMe modifications, in particular adenosine and guanosine, hadadverse affects on knockdown.

DISCUSSION

Using two siRNAs with known immunostimulatory potentials (see Judgeetat, 2005, Nat Biotechnol 23, 457-62), applicant conducted a systematiccomparison of the impact of 20Me and 2′F modifications of guide andpassenger strands on siRNA mediated immune stimulation. As reportedpreviously, applicant found that cytidine modifications were ineffectiveat significantly abrogating the RNA-mediated immunestimulation asmeasured by TNF-alpha induction in human peripheral blood monocytes(Tables 14-17).

Uridine 2′OMe modifications were more effective at reducing TNFa levels33 to 77 fold however modification of individual passenger or guidestrands for “control” siRNA still retained significant TNFa induction,reduced only 2 to 7 fold from unmodified (Table 15). Fluoro modificationof individual strands was largely ineffective while 2′F modification ofboth strands reduced but did not completely abrogate TNFa levels. 2′Furidine modifications have been reported to abrogate cytokine inductionbut this was in the context of a single stranded RNA of differentsequence which may explain the differing results (see Sioud et al.,2007, Biochem Biophys Res Commun, 361, 122-6). Modification ofauanosines had similar results to that observed for uridine with thenotable exception that 2′F guanosines seemed even less effective atreducing TNFa levels (Tables 1447).

In contrast with the other nucleotides, both 2′OMe and 2′F modificationsof adenosine resulted in a striking reduction of TNFa levels (Tables14-16). This effect was pronounced even for modifications of individualstrands and was consistent across both siRNA sequences tested. 2′OMeadenosines reduced TNFa induction ˜50-100 fold whether present on bothstrands or limited to either passenger or guide strands (Table 14),Uridine or guanosine 2′OMe modifications reduced TNFa levels 15-70 foldbut their immunosuppression was less consistent across strands orsequence than the effect observed for 2′OMe adenosine (Table 14). 2′Fadenosine was less effective at reducing TNFa. levels than 2′01Vie (mostevident for B-gal 728) however the suppression of RNA mediated immuneresponse was still highly significant. Furthermore, even the limited 2′For 20Me modification of just three adenosines effectively reduced immunestimulation, in marked contrast to the lack of immune stealth observedwith an equivalent number of uridine modifications (Table 15).

2.′modifications are more broadly tolerated in the siRNA guide strandand have a less deleterious effect on siRNA knockdown activity thancomparable 2′OMe modifications (Table 16). This suggests that 2′Fmodifications, and especially 2′F modified adenosines, are ideallysuited for abrogation of immune stimulation while retaining activity ofsiRNA knockdown. Thus the immune stealth conferred by 2′F adenosinescoupled with their broad tolerance in maintaining RNAi knockdownactivity highlights the value of incorporating 2′F adenosinemodifications into siRNA designs.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein, as presently representative ofpreferred embodiments, are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

I. Tables

TABLE 1 Target SEQ SEQ Site ID Target Modified ID R-Number (human) NO:Sequence Sequence NO: R-007887972-001B 9514 1 CUUUAACAAUUCCUGAAAUB cuuuAAcAAuuccuGAAAuTT B 3 R-007887972-001B 9514 1 CUUUAACAAUUCCUGAAAUAUUucAGGAAuuGuuAAAGUU 4 R-008039792-004D 9514 1 CUUUAACAAUUCCUGAAAUAUUucAGGAAuuGuuAAAGUU 4 R-008039792-004D 9514 1 CUUUAACAAUUCCUGAAAULB cuuuAAcAAuuccuGAAAuTT B 5 R-008245590-000A 291 2 ACAACAGACUUUAAUGUAALB AcAAcAGAcuuuAAuGuAATsT B 6 R-008245590-000A 291 2 ACAACAGACUUUAAUGUAAUUAcAuuAAAGucuGuuGuUsU 7 R-008245595-000U 9514 1 CUUUAACAAUUCCUGAAAULB cuuuAAcAAuucguGAAAuTsT B 8 R-008245595-000U 9514 1CUUUAACAAUUCCUGAAAU AUUucAGGAAuuGuuAAAGUsU 9 R-008276371-000S 9514 1CUUUAACAAUUCCUGAAAU LB cuuuAAcAAuuccuGAAAuTsT B 8 R-008276371-000S 95141 CUUUAACAAUUCCUGAAAU AsUsUsucAGGAAuuGuuAAAGUsU 10 R-008277560-000E 95141 CUUUAACAAUUCCUGAAAU B CUUUaaCaaUUCCUgaaaU TsT B 11 R-008277560-000E9514 1 CUUUAACAAUUCCUGAAAU AsUsUsUCaggaaUUguUaaagUsU 12 R-008277564-000P9514 1 CUUUAACAAUUCCUGAAAU AsUsUsUCaggaaUUguUaaagUsU 12 R-008277564-000P9514 1 CUUUAACAAUUCCUGAAAU LB CUUUaaCaaUUCCUaaaaU TsT B 13R-008298973-000K 291 2 ACAACAGACUUUAAUGUAA LB aCaaCagaCUUUaaUgUaaTsT B14 R-008298973-000K 291 2 ACAACAGACUUUAAUGUAA UsUsAsCaUUaaacUCugUUsUUsU15 R-008308489-0000 291 2 ACAACAGACUUUAAUGUAALB aCaaCagaCUUUaaUsUaaTsT B 14 R-008308489-000G 291 2ACAACAGACUUUAAUGUAA uuaCaUUaaaaUCugUUgUUsU 16 R-008308490-000W 291 2ACAACAGACUUUAAUGUAA LB aCaaCagaCUUUaaUgUaaTsT B 14 R-008308490-000W 2912 ACAACAGACUUUAAUGUAA uuACaUUaagUCugUUgUUsU 17 R-008313344-000A 9514 1CUUUAACAAUUCCUGAAAU LB CUUUaaCaaUUCCUgaaaU TsT B 13 R-008313344-000A9514 1 CUUUAACAAUUCCUGAAAU AuUUCaggaaUUguUaaagUsU 18 R-008313345-000J9514 1 CUUUAACAAUUCCUGAAAU LB CUUUaaCaaUUCCUgaaaU TsT B 13R-008313345-000J 9514 1 CUUUAACAAUUCCUGAAAU auuUCaggaaUUguUaaagUsU 19R-008313350-000H 9514 1 CUUUAACAAUUCCUGAAAU LB CUUUaaCaaUUCCUgaaaU TsT B13 R-008313350-000H 9514 1 CUUUAACAAUUCCUGAAAU AuT UCaggaaUUguUaaagUsU20 R-008313356-000K 9514 1 CUUUAACAAUUCCUGAAAU LB CUUUaaCaaUUCCUgaaaUTsT B 13 R-008313356-000K 9514 1 CUUUAACAAUUCCUGAAAU auTUCaggaaUUguUaaagUsU 21 R-008333359-000l 291 2 ACAACAGACUUUAAUGUAALB aCaaCagaCUUUaaUgUaaTsT B 14 R-008313359-000L 291 2ACAACAGACUUUAAUGUAA TuACaUUaaagUCugUUgUUsU 22 R-008313361-000J 291 2ACAACAGACUUUAAUGUAA LB aCaaCagaCUUUaaUgUaaTsT B 14 R-008313361-000J 2912 ACAACAGACUUUAAUGUAA TuACaUUaaagUCUsUUgUUsU 23 R-008277563-00GX 9514 1CUUUAACAAUUCCUGAAAU AsUsUsCaggaaUUaAagUsU 12 R-008277562-000X 9514 1CUUUAACAAUUCCUGAAAU LB CUUUAACAAUUCCUGAAAU TsT B 24 R-008290704-000W9514 1 CUUUAACAAUUCCUGAAAU LB CUUUAACAAUUCCUGAAAU TsT B 24R-008290704-000W 9514 1 CUUUAACAAUUCCUGAAAU AsUsUsUCAGGAAUUGuUAAAGTsT 25R-008347773-000D 291 2 ACAACAGACUUUAAUGUAA LB ACAACAGACUUUAAUGUAATsT B26 R-008347773-000D 291 2 ACAACAGACUUUAAUGUAA CuACAUUAAAGUCuGUUGU TsT 27R-008347763-000L 291 2 ACAACAGACUUUAAUGUAA LB ACAACAGACUUUAAUGUAATsT B26 R-008347763-000L 291 2 ACAACAGACUUUAAUGUAA uuACAUUAAAGUCuGUUGUTsT 28R-008357258-000C 19 31 CUCUCACAUACAAUUGAAA B CUCUCACAUACAAUUGAAAUsU B111 R-008357258-000C 19 31 CUCUCACAUACAAUUGAAA UUUCAAUUGUAUGUGAGAGUsU112 R-008357080-000R 248 32 CAGUCCUGAAGGAAUCCAUB CAGUCCUGAAGGAAUCCAUUsU B 113 R-008357080-000R 248 32CAGUCCUGAAGGAAUCCAU AUGGAUUCCUUCAGGACUGUsU 114 R-008355914-000C 397 33GGUAUGACUGUCAAAGUAA B GGUAUGACUGUCAAAGUAAUsU B 115 R-008355914-000C 39733 GGUAUOACUGUCAAAGUAA UUACUUUGACAGUCAUACCUsU 116 R-008356933-000Y 48534 CCAGUAAGGCUUCUCUUAA B CCAGUAAGGCUUCUCUUAAUsU B 117 R-008356933-000Y485 34 CCAGUAAGGCUUCUCUUAA UUAAGAGAAGCCUUACUGGUsU 118 R-008356751-000W601 35 GGCAUACAUUCGUCCCAAA B GGCAUACAUUCGUCCCAAAUsU B 119R-008356751-000W 601 35 GGCAUACAUUCGUCCCAAA UUUCXXiACGAAUGUAUGCCUsU 120R-008355911-000B 719 36 GCUUCCUCAACUAUUCUAA B GCUUCCUCAACUAUUCUAAUsU B121 R-008355911-000B 719 36 GCUUCCUCAACUAUUCUAA UUAGAAUAGUUGAGGAAGCUsU122 R-008356343-000G 780 37 CAGCAUUCUAACAGCCAAUB CAGCAUUCUAACAGCCAAUUsU B 323 R-008356343-000G 780 37CAGCAUUCUAACAGCCAAU AUUGGCUGUUAGAAUGCUGUsU 124 R-008357252-000A 1124 38GUAUAGGAAUGAAUGGAGA B GUAUAGGAAUGAAUGGAGAUsU B 125 R-008357252-000A 112438 GUAUAGGAAUGAAUGGAGA UCUCCAUUCAUUCCUAUACUsU 126 R-008356340-000F 144539 CCUCCUAUAAUGAAGCAAA B CCUCCUAUAAUGAAGCAAAUsU B 127 R-008356340-000F1445 39 CCUCCUAUAAUGAAGCAAA UUUGCUUCAUUAUAGGAGGUsU 128 R-008357255-000B1446 40 CUCCUAUAAUGAAGCAAAA B CUCCUAUAAUGAAGCAAAAUsU B 129R-008357255-000B 1446 40 CUCCUAUAAUGAAGCAAAA UUUUGCUUCAUUAUAGGAGUsU 130R-008356337-000Z 1983 41 CUCUCUAACUAACAAAUUU B CUCUCUAACUAACAAAUUUUsU B131 R-008356337-000Z 1983 41 CUCUCUAACUAACAAAUUU AAAUUUGUUAGUUAGAGAGUsU132 R-008355917-000D 3214 42 CAAGCAGAAGGAGUGCAGCB CAAGCAGAAGGAGUGCAGCUsU B 133 R-008355917-000D 3214 42CAAGCAGAAGGAGUGCAGC GCUGCACUCCUUCUGCUUGUsU 134 R-008357077-000J 3614 43AUGAGAUAAUAGAAUUUGA B AUGAGAUAAUAGAAUUUGAUsU B 135 R-008357077-000J 361443 AUGAGAUAAUAGAAUUUGA UCAAAUUCUAUUAUCUCAUUsU 136 R-008356128-000W 454244 CGUCAAAGAUAUCAAGGUU B CGUCAAAGAUAUCAAGGUUUsU B 137 R-008356128-000W4542 44 CGUCAAAGAUAUCAAGGUU AACCUUGAUAUCUUUGACGUsU 138 R-008356561-000U6548 45 GAAUUACAGAUAAUGAUGU B GAAUUACAGAUAAUGAUGUUsU B 139R-008356561-000U 6548 45 GAAUUACAGAUAAUGAUGU ACAUCAUUAUCUGUAAUUCUsU 140R-008355905-000U 6930 46 CAUUCAGCAGCUUGCUGCA B CAUUCAGCAGCUUGCUGCAUsU B141 R-008355905-000U 6930 46 CAUUCAGCAGCUUGCUGCA UGCAGCAAGCUGCUGAAUGUsU142 R-008356558-000M 6981 47 CACAAUGCAUUUAGAUCAAB CACAAUGCAUUUAGAUCAAUsU B 143 R-008356558-000M 6981 47CACAAUGCAUUUAGAUCAA UUGAUCUAAAUGCAUUGUGUsU 144 R-008357083-000S 7044 48CCGUGUCAAAUACUUUGUU B CCGUGUCAAAUACUUUGUUUsU B 145 R-008357083-000S 7044148 CCGUGUCAAAUACUUUGUU AACAAAGUAUUUGACACGGUsU 146 R-008356334-000Y 941449 CAUAGAAGCCAGUAUAGGA B CAUAGAAGCCAGUAUAGGAUsU B 147 R-008356334-000Y9454 49 CAUAGAAGCCAGUAUAGGA UCCUAUACUGGCUUCUAUGUsU 148 R-008357249-000U9514 1 CUUUAACAAUUCCUGAAAU B CUUUAACAAUUCCUGAAAUsU B 149R-008357249-000U 9514 1 CUUUAACAAUUCCUGAAAU AUUUCAGGAAUUGUUAAAGUsU 150R-008356555-000L 9621 1 ACAAAGCAAUCAUUUGAUU B ACAAAGCAAUCAUUUGAUUUsU B151 R-008356555-000L 9621 50 ACAAAGCAAUCAUUUGAUU AAUCAAAUGAUUGCUUUGUUsU152 R-008356930-000X 10162 51 CAAGUGUCAUCACACUGAAB CAAGUGUCAUCACACUGAAUsU B 153 R-00835693G-000X 10162 51CAAGUGUCAUCACACUGAA UUCAGUGUGAUGACACUUGUsU 154 R-008356552-000K 10167 52GUCAUCACACUGAAUACCA B GUCAUCACACUGAAUACCAUsUU 155 R-008356552-000K 1016752 GUCAUCACACUGAAUACCA UGGUAUUCAGUGUGAUGACUsU 156 R-008356331-000X 1016853 UCAUCACACUGAAUACCAA B UCAUCACACUGAAUACCAAUsU B 157 R-008356331-000X10168 53 UCAUCACACUGAAUACCAA UUGGUAUUCAGUGUGAUGAUsU 158 R-008356125-000V10219 54 CAGUACAAAUUAGAGGGAA B CACUACAAAUUAGAGGGAAUsU B 159R-008356125-000V 10219 54 CAGOACAAAUUAGAGGGAA UUCCCUCUAAUUUGUACUGUsU 160R-008356549-000D 10455 55 GAACUUAAUGGAAAUACCA B GAACUUAAUGGAAAUACCAUsU B161 R-008356549-000D 10455 55 GAACUUAAUGGAAAUACCA UGGUAUUUCCAUUAAGUUCUsU162 R-008356329-000Z 10517 56 UUGAUCACAAGUUCAGCUUB UUGAUCACAAGUUCAGCUUUsU B 163 R-008356329-000Z 10517 56UUGAUCACAAGUUCAGCUU AAGCUGAACUUGUGAUCAAUsU 164 R-008356326-000Y 12673 57GAGAAAUCAAGAUUAAUCA B GAGAAAUCAAGAUUAAUCAUsU B 165 R-008356326-000Y12673 57 GAGAAAUCAAGAUUAAUCA UGAUUAAUCUUGAUUUCUCUsU 166 R-008356748000P13666 58 CUUUGUAGACUACUAUAAA B CUUUGUAGACUACUAUAAAUsU B 167R-008356748-000P 13666 58 CUUUGUAGACUACUAUAAA UUUAUAGUAGUCUACAAAGUsU 168R-008355979-000A 19 31 CUCUCACAUACAAUUGAAA B CUCUCaCaUaCaaUUcaaaTsT B169 R-008355979-000A 19 31 CUCUCACAUACAAUUGAAA UsUsUsCaaUUgUaUgucagagUsU170 R-008356396-000V 248 32 CAGUCCUGAAGGAAUCCAU B CaaUCCUaaaggaaUCCaUTsT B 171 R-008356396-000V 248 32 CAGUCCUGAAGGAAUCCAUAsUsGsgaUUCCUUCaggaCUgUsU 172 R-008357291-000L 397 33GGUAUGACUGUCAAAGUAA B ggUaUgaCUaUCaaagUaaTsT B 173 R-008357291-000L 39733 GGUAUGACUGUCAAAGUAA UsUsUsCUUUgaCagUcaUaCCUsU 174 R-008357122-000N485 34 CCAGUAAGGCUUCUCUUAA B CCacUaaggCUUCUCUUaaTsT B 175R-008357122-000N 485 34 CCAGUAAGGCUUCUCUUAA UsUsAsagagaagCCUuaCUggUsU176 R-008355976-000Z 601 35 GGCAUACAUUCGUCCCAAAB ggCaUaCaUUCgUCCCaaaTsT B 177 R-008355976-000Z 601 35GGCAUACAUUCGUCCCAAA UsUsUsgggaCgaaUguaUgCCUsU 178 R-008357288-000E 71936 GCUUCCUCAACUAUUCUAA B aCUUCCUCaaCUaUUCUaaTsT B 179 R-008357288-000E719 36 GCUUCCUCAACUAUUCUAA UsUsAsgaaUagUUgaggaagCUsU 180R-008356569-000N 780 37 CAGCAUUCUAACAGCCAAU B CagCaUUCUaaCagCCaaU TsT B181 R-008356569-000N 780 37 CAGCAUUCUAACAGCCAAUAsUsUsggCUgUUagaaUgCUgUsU 182 R-008356393-000U 1124 38GUAUAGCAAUGAAUGGAGA B gUaUaggaaUgaaUggagaTsT B 183 R-008356393-000U 112438 GUAUAGGAAUGAAUGGAGA UsCsUsCCaUUCaUUCcUaUaCUsU 184 R-008355973-000Y1445 39 CCUCCUAUAAUGAAGCAAA B CCUCCUaUaaUgaagCaaaTsT B 185R-008355973-000Y 1445 39 CCUCCUAUAAUGAAGCAAA UsUsUsgCUUCaUUaUaggaggUsU186 R-008356941-000Y 1446 40 CUCCUAUAAUGAAGCAAAAB CUCCUaUaaUgaagCaaaaTsT B 187 R-008356941-000Y 1446 40CUCCUAUAAUGAAGCAAAA UsUsUsUgCUUCaUUauaggsagUsU 188 R-008356184-000R 198341 CUCUCUAACUAACAAAUUU B CUCUCUaaCUaaCaaaUUU TsT B 189 R-008356184-000R1983 41 CUCUCUAACUAACAAAUUU AsAsAsUUUgUUagUUagagagUsU 190R-008356351-000G 3714 42 CAAGCAGAAGGAGUGCAGC B CaagCagaaggagUgCagC TsT B191 R-008356351-000G 3314 42 CAAGCAGAAGOAGUGCACCGsCsUsgCaCUCCUUCucCUUgUsU 192 R-008356795-000A 3614 43AUGAGAUAAUAGAAUUUGA B aUgagaUaaUagaaUUUgaTsT B 193 R-008356795-000A 361443 AUGAGAUAAUAGAAUUUGA UsCsAsaaUUCUaUUauCUCaUUsU 194 R-008356604-000A4542 44 CGUCAAAGAUAUCAAGGUU B CgUCaaagaUaUCaaggUU TsT B 195R-008356604-000A 4542 44 CGUCAAAGAUAUCAAGGUU AsAsCsCUUgaUaUCUaUgaCgUsU196 R-008356134-000D 6548 45 GAAUUACAGAUAAUGAUGUB gaaUUaCagaUaaUgaUgUTsT B 197 R-008356134-000D 6548 45GAAUUACAGAUAAUGAUGU AsCsAsUCaUUaUCUguaUUCUsU 198 R-008357119-0000 693046 CAUUCAGCAGCUUGCUGCA B CaUUCagCagCUUgCUgCaTsT B 199 R-008357119-000G6930 46 CAUUCAGCAGCUUGCUGCA UsGsCsagCaagCUcCugaaUgUsU 200R-008356181-000P 6981 47 CACAAUGCAUUUAGAUCAA B CaCaaUaCaUUUacaUCaaTsT B201 R-008356181-000P 6981 47 CACAAUGCAUUUAGAUCAAUsUsGsaUCUaaaUgCaUUgUgUsU 202 R-008355923-000L 7044 48CCGUGUCAAAUACUUUGUU B CCgUgUCaaaUaCUUUgUU TsT B 203 R-008355923-000L7044 48 CCGUGUCAAAUACUUUGUU AsAsCsaaagUaUUUgaCaCscUsU 204R-008356969-000C 9414 49 CAUAGAAGCCAGUAUAGGA B CaUaaaagCCaaUaUaggaTsT B205 R-008356969-000C 9414 49 CAUAGAAGCCAGUAUAGGAUsCsCsUaUaCUggCUuCUaUaUsU 206 R-008356767-000R 9621 50ACAAAGCAAUCAUUUGAUU B aCaaagCaaUCaUUUsaUU TsT B 207 R-008356767-000R9621 50 ACAAAGCAAUCAUUUGAUU AsAsUsCaaaUcaUUsgUUUcUUsU 208R-008356601-000Z 10162 51 CAAGUGUCAUCACACUGAA B CaaaUsUCaUCaCaCUgaaTsT B209 R-008356601-000Z 10162 51 CAAGUGUCAUCACACUGAAUsUsCsagUgUgaUgacaCUUgUsU 210 R-008356598-0000 10167 52GUCAUCACACUGAAUACCA B gUCaUCaCaCUgaaUaCCaTsT B 211 R-008356598-000G10167 52 GUCAUCACACUGAAUACCA UsGsGsUaUUCagUgUgaUgaCUsU 212R-008279809-000X 10168 53 UCAUCACACUGAAUACCAA B UCaUCaCaCUgaaUaCCaaTsT B213 R-008279809-000X 10168 53 UCAUCACACUGAAUACCAAUsUsGsgUaUUCaaUgugaUgaUsU 214 R-008355970-000X 10219 54CAGUACAAAUUAGAGGGAA B CagUaCaaaUUagagggaaTsT B 215 R-008355970-000X10219 54 CAGUACAAAUUAGAGGGAA UsUsCsCCUCUaaUUUgUaCUaUsU 216R-008355967-000R 10455 55 GAACUUAAUGGAAAUACCA B gaaCUUaaUggaaaUaCCaTsT B217 R-008355967-000R 10455 55 GAACUUAAUGGAAAUACCAUsGsGsUaUUUCCaUUaagUUCUsU 218 R-008356178-000H 10517 56UUGAUCACAAGUUCAGCUU B UUgaUCaCaagUUCagCUU TsT B 219 R-008356178-000H10517 56 UUGAUCACAAGUUCAGCUU AsAsGsCUaaaCUUaUsaUCaaUsU 220R-008356792-000Z 12673 57 GAGAAAUCAAGAUUAAUCA B gagaaaUCaagaUUaaUCaTsT B221 R-008356792-000Z 12673 57 GAGAAAUCAAGAUUAAUCAUsGsAsUUaaUCUUaauUUCUCUsU 222 R-008356387-000L 13666 58CUUUGUAGACUACUAUAAA B CUUUsUagaCUaCUaUaaaTsT B 223 R-008356387-000L13666 58 CUUUGUAGACUACUAUAAA UsUsUsaUagUagUCUaCaaasUsU 224R-008391240-000E 70 59 CGUUGAUAACCCAAAUGGA B CGUUGAUAACCCAAAUGGAUsU B225 R-008391240-000E 70 59 CGUUGAUAACCCAAAUGGA UCCAUUUGGGUUAUCAACGUsU226 R-008391213-000D 93 60 IGAAGAUGCGUGACAUGUAUB GAAGAUGCGUGACAUGUAUUsU B 227 R-008391213-000D 93 60GAAGAUGCGUGACAUGUAU AUACAUGUCACGCAUCUUCUsU 228 R-008313809-000Y 146 61AGUGGAGGUAUUCUUCGAA B AGUGGAGGUAUUCUUCGAAUsU B 229 R-008313809-000Y 14661 AGUGGAGGUAUUCUUCGAA UUCGAAGAAUACCUCCACUUsU 230 R-008313864-000J 19662 CAUUGAACCCAAAUUUGAU B CAUUGAACCCAAAUUUGAUUsU B 231 R-008313864-000J196 62 CAUUGAACCCAAAUUUGAU AUCAAAUUUGGGUUCAAUGUsU 232 R-008391328-00GZ284 63 OCAAUAACUGUUUGGUAUU B GCAAUAACUGUUUGGUAUUUsU B 233R-008391328-000Z 284 63 GCAAUAACUGUUUGGUAUU AAUACCAAACAGUUAUUGCUsU 234R-008391263-000R 384 64 CAGUCAGCAAAGACGUCUA B CAGUCAGCAAAGACGUCUAUsU B235 R-008391263-000R 384 64 CAGUCAGCAAAGACGUCUA UAGACGUCUUUGCUGACUGUsU236 R-008391207-000W 420 65 GCAGUACCCACGUCACCUAB GCAGUACCCACGUCACCUAUsU B 237 R-008391207-000W 420 65GCAGUACCCACGUCACCUA UAGGUGACGUGGGUACUGCUsU 238 R-008391296-000U 485 66GAGACACCUGCCUGGUAUU B GAGACACCUGCCUGGUAUUUsU B 239 R-008391296-000U 48566 GAGACACCUGCCUGGUAUU AAUACCAGGCAGGUGUCUCUsU 240 R-008391228-000P 66167 GAAACAAGGGCCCUUUGUA B GAAACAAGGGCCGUUUGUAUsU B 241 R-008391228-000P661 67 GAAACAAGGGCCCUUUGUA UACAAAGGGCCCUUGUUUCUsU 242 R-008391414-000G780 68 CAAGGACCCCGGCUGCGAA B CAAGGACCCCGGCUGCGAAUsU B 243R-008391414-000G 780 68 CAAGGAGCCCGGCUGGGAA UUCGCAGCCGGGCUCCUUGUsU 244R-008391414-000F 849 69 CGGGAAGCUGGGCAGCUAC B CGGGAAGCUCUGGGCAGCUACUsU B245 R-008391411-000F 849 69 CGGGAAGCUGGGCAGCUAC GUAGCUGCCCAGCUUCCCGUsU246 R-008391314-000X 881 70 GGACGAAAGCCAUGGUUGCB GCTACGAAAGCCAUGGUUGCUsU B 247 R-008391314-000X 881 70GGACGAAAGCCAUGGUUGC GCAACCAUGGCUUUCGUCCUsU 248 R-008391325-000Y 887 71AAGCCAUGGUUGCUUGUUA B AAGCCAUGGUUGCUUGUUAUsU B 249 R-008391325-000Y 88771 AAGCCAUGGUUGCUUGUUA UAACAAGCAACCAUGGCUUUsU 250 R-008350794-C00Z 95572 GAUGGAAGAUGUGUGACAU B GAUGGAAGAUGUGUGACAUUsU B 251 R-0G8350794-000Z955 72 GAUGGAAGAUGUGUGACAU AUGUCACACAUCUUCCAUCUsU 252 R-008350713-000B962 73 GAUGUGUGACAUGUAUAUA B GAUGUGUGACAUGUAUAUAUsU B 253R-008350713-000B 962 73 GAUGUGUGACAUGUAUAUA UAUAUACAUGUCACACAUCUsU 254R-008391266-000S 994 74 GACUGGGAUGCCAAGGUAA B GACUGGGAUGCCAAGGUAAUsU B255 R-008391266-000S 994 74 GACUGGGAUGCCAAGGUAA UUACCUUGGCAUCCCAGUCUsU256 R-008391357-000T 1048 75 GCCCAGUUUGCUGACAUUGB GCCCAGUUUGCUGACAUUGUsU B 257 R-008391357-000T 1048 75GCCCAGUUUGCUGACAUUG CAAUGUCAGCAAACUGGGCUsU 258 R-008391234-000X 1055 76UUGCUGACAUUGAACCCAA B UUGCUGACAUUGAACCCAAUsU B 259 R-008391234-000X 105576 UUGCUGACAUUGAACCCAA UUGGGUUCAAUGUCAGCAAUsU 260 R-008391302-000M 110777 UCGCAACCCUCAUGAAGUA B UCGCAACCCUCAUGAAGUAUsU B 261 R-008391302-000M1107 77 UCGCAACCCUCAUGAAGUA UACUUCAUGAGGGUUGCGAUsU 262 R-008391299-000V1115 78 CUCAUGAAGUACAACCACC B CUCAUGAAGUACAACCAGCUsU B 263R-008391299-000V 1115 78 CUCAUGAAGUACAACCAGC GCUGGUUGUACUUCAUGAGUsU 264R-008391354-000S 1223 79 GUGUGAGGGUUGAACUCAA B GUGUGAGGGUUGAACUCAAUsU B265 R-008391354-000S 1223 79 GUGUGAGGGUUGAACUCAA UUGAGUUCAACCCUCACACUsU266 R-008313818-000G 4295 80 AUGCUACAAGGUACGCAAUB AUGCUACAAGGUACGCAAUUsU B 267 R-008313818-000G 4295 80AUGCUACAAGGUACGCAAU AUUGCGUACCUUGUAGCAUUsU 268 R-008313815-000F 4302 81AAGGUACGCAAUAACUGUU B AAGGUACGCAAUAACUGUUUsU B 269 R-008313815-000F 430281 AAGGUACGCAAUAACUGUU AACAGUUAUUGCGUACCUUUsU 270 R-008391381-000T 438182 GGUGUGAGGGUUGAACUCA B GGUGUGAGGGUUGAACUCAUsU B 271 R-008391381-000T4381 82 GGUGUGAGGGUUGAACUCA UGAGUUCAACCCUCACACCUsU 272 R-008391351-000R70 59 CGUUGAUAACCCAAAUGGA B CgUUgaUaaCCCaaaUggaTsT B 273R-008391351-000R 70 59 CGUUGAUAACCCAAAUGGA UsCsCsaUUUscaUUauCaaCgUsU 274R-008391293-000T 93 60 GAAGAUGCGUGACAUGUAU B gaagaUgCgUgaCaUgUaU TsT B275 R-008391293-000T 93 60 GAAGAUGCGUGACAUGUAU AsUsAsCaUgUCaCgCaUCUUCUsU276 R-008391258-000S 146 61 AGUGGAGGUAUUCUUCGAAB agUggaggUaUUCUUCgaaTsT B 277 R-008391258-000S 146 61AGUGGAGGUAUUCUUCGAA UsUsCsgaagaaUaCCuCCaCUUsU 278 R-008391290-000S 19662 CAUUGAACCCAAAUUUGAU B CaUUgaaCCCaaaUUUsaU TsT B 279 R-008391290-000S196 62 CAUUGAACCCAAAUUUGAU AsUsCsaaaUUUacaUuCaaUgUsU 280R-008391372-000J 284 63 GCAAUAACUGUUUGGUAUU B gCaaUaaCUgUUUagUaUU TsT B281 R-008391372-000J 284 63 GCAAUAACUGUUUGGUAUUAsAsUsaCCaaaCagUuaUUgCUsU 282 R-008391348-000J 384 64CAGUCAGCAAAGACGUCUA B CagUCagCaaagaCgUCUaTsT B 283 R-008391348-000J 38464 CAGUCAGCAAAGACGUCUA UsAsGsaCgUCUUUaCugaCUgUsU 284 R-008391287-000K420 65 GCAGUACCCACGUCACCUA B gCagUaCCCaCcUCaCCUaTsT B 285R-008391287-000K 420 65 GCAGUACCCACGUCACCUA UsAsGsgUgaCgUggguaCUgCUsU286 R-008391345-000H 485 66 GAGACACCUGCCUGGUAUU  B aagaCaCCUgCCUagUaUUTsT B 2S7 R-008391345-000H 485 66 GAGACACCUGCCUGGUAUUAsAsUsaCCaaaCaaguaUCUCUsU 288 R-008391311-000W 661 67GAAACAAGGGCCCUUUGUA B gaaaCaagggCCCUUUgUaTsT B 289 R-008391311-000W 66167 GAAACAAGGGCCCUUUGUA UsAsCsaaagggCCCUugUUUCUsU 290 R-008391369-000C780 68 CAAGGAGCCCGGCUGCGAA B CaaagaaCCCggCUgCgaaTsT B 291R-008391369-000C 780 68 CAAGGAGCCCGGCUGCGAA UsUsCsaCaaCCaagCuCCUUgUsU292 R-008391342-000G 849 69 CGGGAAGCUGGGCAGCUAC B CgggaaaCUgsgCagCUaCTsT B 293 R-008391342-000G 849 69 CGGGAAGCUGGGCAGCUACGsUsAsgCUgCCCagCuUCCCgUsU 294 R-008391366-000B 881 70GGACGAAAGCCAUGGUUGC B ggaCgaaagCCaUggUUgC TsT B 295 R-008391366-000B 88170 GGACOAAAGCCAUGGUUGC GsCsAsaCCaUggCUUuCgUCCUsU 296 R-008391405-000Y887 71 AAGCCAUGGUUGCUUGUUA B aacCCaUggUUgCUUgUUaTsT B 297R-008391405-000Y 887 71 AAGCCAUGGUUGCUUGUUA UsAsAsCaagCaaCCauggCUUUsU298 R-008391255-000R 955 72 GAUGGAAGAUGUGUGACAU B gaUggaagaUgUgUgaCaUTsT B 299 R-008391255-000R 955 72 GAUGGAAGAUGUGUGACAUAsUsGsUCaCaCaUCUuCCaUCUsU 300 R-008391402-000X 962 73GAUGUGUGACAUGUAUAUA B gaUgUgUgaCaUgUaUaUaTsT B 301 R-008391402-000X 96273 GAUGUGUGACAUGUAUAUA UsAsUsaUaCaUgUCacaCaUCUsU 302 R-008391192-000Z994 74 GACUGGGAUGCCAAGGUAA B gaCUgggaUgCCaaggUaaTsT B 303R-008391192-000Z 994 74 GACUGGGAUGCCAAGGUAA UsUsAsCCUUgaCaUCcCacUCUsU304 R-008391284-000J 1048 75 GCCCAGUUUGCUGACAUUGB gCCCagUUUgCUgaCaUUgTsT B 305 R-008391284-000J 1048 75GCCCAGUUUGCUGACAUUG CsAsAsUgUCagCaaacUgggCUsU 306 R-008391281-000H 105576 UUGCUGACAUUGAACCCAA B UUgCUgaCaUUgaaCCCaaTsT B 307 R-008391281-000H1055 76 UUGCUGACAUUGAACCCAA UsUsGsggUUCaaUgUcagCaaUsU 308R-008391201-000U 1107 77 UCGCAACCCUCAUGAAGUA B UCgCaaCCCUCaUgaagUaTsT B309 R-008391201-000U 1107 77 UCGCAACCCUCAUGAAGUAUsAsCsUUCaUgacGauUgCgaUsU 310 R-008391252-000P 1115 78CUCAUGAAGUACAACCAGC B CUCaUgaaUUaCaaCCagC TsT B 311 R-008391252-000P1115 78 CUCAUGAAGUACAACCAGC GsCsUsggUUgUaCUUcaUgagUsU 312R-008391198-000B 1223 79 GUGUGAGGGUUGAACUCAA B gUgUgagggUUgaaCUCaaTsT B313 R-008391198-000B 1223 79 GUGUGAGGGUUGAACUCAAUsUsGsagUUCaaCCCuCaCaCUsU 314 R-008391249-000H 4295 80AUGCUACAAGGUACGCAAU B aUgCUaCaaggUaCsCaaU TsT B 315 R-008391249-000H4295 80 AUGCUACAAGGUACGCAAU AsUsUsgCgUaCCUUguagCaUUsU 316R-008391246-000G 4302 81 AAGGUACGCAAUAACUGUU B aaggUaCgCaaUaaCUgUU TsT B317 R-008391246-000G 4302 81 AAGGUACGCAAUAACUGUUAsAsCsagUUaUUgCguaCCUUUsU 318 R-008391222-000M 4381 82GGUGUGAGGGUUGAACUCA B ggUgUgagggUUgaaCUCaTsT B 319 R-008391222-000M 438182 GGUGUGAGGGUUGAACUCA UsGsAsaUUCaaCCCUcaCaCCUsU 320 R-008357193-000U243 83 UGAAGGCUGGGUACCUUUG B UGAAGGCUGGGUACCUUUGUsU B 321R-008357193-000U 243 83 UGAAGGCUGGGUACCUUUG CAAAGGUACCCAGCCUUCAUsU 322R-008356271-000G 253 84 AUCUGUCAUCAAAUUGAGU B AUCUGUCAUCAAAUUGAGUUsU B323 R-008356271-000G 253 84 AUCUGUCAUCAAAUUGAGU ACUCAAUUUGAUGACAGAUUsU324 R-008356480-000K 254 85 UCUGUCAUCAAAUUGAGUAB UCUGUCAUCAAAUUGAGUAUsU B 325 R-008356480-000K 254 85UCUGUCAUCAAAUUGAGUA UACUCAAUUUGAUGACAGAUsUUsU 326 R-008356688-000Z 25586 CUGUCAUCAAAUUGAGUAU B CUGUCAUCAAAUUGAGUAUUsU B 327 R-008356688-000Z255 86 CUGUCAUCAAAUUGAGUAU AUACUCAAUUUGAUGACAGUUsU 328 R-008357396-C00P257 87 GUCAUCAAAUUGAGUAUUA B GUCAUCAAAUUGAGUAUUAUsU B 329R-008357396-000P 257 87 GUCAUCAAAUUGAGUAUUA UAAUACUCAAUUUGAUGACUsU 330R-008356265-000Z 258 88 UCAUCAAAUUGAGUAUUAU B UCAUCAAAUUGAGUAUUAUUsU B331 R-008356265-000Z 258 88 UCAUCAAAUUGAGUAUUAU AUAAUACUCAAUUUGAUGAUsU332 R-008357199-000W 279 89 UGGAGACUUCAAUUUGCCAB UGGAGACUUCAAUUUGCCAUsU B 333 R-008357199-000W 279 89UGGAGACUUCAAUUUGCCA UGGCAAAUUGAAGUCUCCAUsU 334 R-008356273-000Z 291 2ACAACAGACUUUAAUGUAA B ACAACAGACUUUAAUGUAAUsU B 335 R-0C8356273-000Z 2912 ACAACAGACUUUAAUGUAA UUACAUUAAAGUCUGUUGUUsU 336 R-008356262-000Y 329 90UGGAUGAAGGCUGGGUACC B UGGAUGAAGGCUGGGUACCUsU B 337 R-008356262-000Y 32990 UGGAUGAAGGCUGGGUACC GGUACCCAGCCUUCAUCCAUsU 338 R-008357393-000N 33091 GGAUGAAGGCUGGGUACCU B GGAUGAAGGCUGGGUACCUUsU B 339 R-008357393-000N330 91 GGAUGAAGGCUGGGUACCU AGGUACCCAGCCUUCAUCCUsU 340 R-008357040-000W331 92 GAUGAAGGCUGGGUACCUU B GAUGAAGGCUGGGUACCUUUsU B 341R-008357040-000W 331 92 GAUGAAGGCUGGGUACCUU AAGGUACCCAGCCUUCAUCUsU 342R-008356477-000D 332 93 AUGAAGGCUGGGUACCUUU B AUGAAGGCUGGGUACCUUUUsU B343 R-008356477-000D 332 93 AUGAAGGCUGGGUACCUUU AAAGGUACCCAGCCUUCAUUsU344 R-008356871-000R 335 94 AAGGCUGGGUACCUUUGGAB AAGGCUGGGUACCUUUGGAUsU B 345 R-008356871-000R 335 94AAGGCUGGGUACCUUUGGA UCCAAAGGUACCCAGCCUUUsU 346 R-008357390-000M 337 95GGCUGCGUACCUUUGGAAA B GGCUGGGUACCUUUGGAAAUsU B 347 R-008357390-000M 33795 GGCUGGGUACCUUUGGAAA UUUCCAAAGGUACCCAGCCUsU 348 R-008356060-000L 33996 CUGGGUACCUUUGGAAACA B CUGGGUACCUUUGGAAACAUsU B 349 R-0G8356060-000L339 96 CUGGGUACCUUUGGAAACA UGUUUCCAAAGGUACCCAGUsUUsU 350R-008357196-000V 485 97 GCAGACCACUCCCUGAAGU B GCAGACCACUCCCUGAAGUUsU B351 R-008357196-000V 496 97 GCAGACCACUCCCUGAAGU ACUUCAGGGAGUGGUCUGCUsU352 R-008356057-000E 496 98 CCUGAAGUGACGGAUGAGUB CCUGAAGUGACGGAUGAGUUsU B 353 R-008356057-000E 496 98CCUGAAGUGACGGAUGAGU ACUCAUCCGUCACUUCAGGUsU 354 R-008356275-000S 869 99AAAUCAUGGUGAAAUAAAA B AAAUCAUGGUGAAAUAAAAUsU B 355 R-008356275-000S 86999 AAAUCAUGGUGAAAUAAAA UUUUAUUUCACCAUGAUUUUsU 356 R-008357190-000T 1065100 UGCAAAUAAUGGUAACCUA B UGCAAAUAAUGGUAACCUAUsU B 357 R-008357190-000T1065 100 UGCAAAUAAUGGUAACCUA UAGGUUACCAUUAUUUGCAUsU 358 R-008357037-000P1066 101 GCAAAUAAUGGUAACCUAC B GCAAAUAAUGGUAACCUACUsU B 359R-008357037-0G0P 1066 101 GCAAAUAAUGGUAACCUAC GUAGGUUACCAUUAUUUGCUsU 360R-008356483-000L 1070 102 AUAAUGGUAACCUACUGUU B AUAAUGGUAACCUACUGUUUsU B361 R-008356483-000L 1070 102 AUAAUGGUAACCUACUGUU AACAGUAGGUUACCAUUAUUsU362 R-008356259-000S 1075 103 GGUAACCUACUGUUAAGGAB GGUAACCUACUGUUAAGGAUsU B 363 R-008356259-000S 1075 103GGUAACCUACUGUUAAGGA UCCUUAACAGUAGGUUACCUsU 364 R-008356682-000X 1112 104AAGUACUAGAAGGACAUGC B AAGUACUAGAAGGACAUGCUsU B 365 R-008356682-000X 1112104 AAGUACUAGAAGGACAUGC GCAUGUCCUUCUAGUACUUUsU 366 R-008356278-000T 1304105 AAGGAAGAGGACAGUUUCA B AAGGAAGAGGACAGUUUCAUsU B 367 R-008356278-000T1304 105 AAGGAAGAGGACAGUUUCA UGAAACUGUCCUCUUCCUUUsU 368 R-008356054-000D1328 106 GGAGGACAAGAUUUGAUGA B GGAGGACAAGAUUUGAUGAUsU B 369R-008356054-000D 1328 106 GGAGGACAAGAUUUGAUGA UCAUCAAAUCUUGUCCUCUsU 370R-008356471-000B 1395 107 CAGAGAAGAACCCGCAUCA B CAGAGAAGAACCCGCAUCAUsU B371 R-008356471-000B 1395 107 CAGAGAAGAACCCGCAUCA UGAUGCGGGUUCUUCUCUGUsU372 R-008357387-000F 1397 108 GAGAAGAACCCGCAUCAAA B GAGAAGAACCCGCAUCAAAUsU B 373 R-008357387-000F 1397 108GAGAAGAACCCGCAUCAAA U UUGAUGCGGGUUCUUCUCUsU 374 R-008357450-000C 243 83UGAAGGCUGGGUACCUUUG B UgaaggCUgggUaCCUUUgTsT B 375 R-008357450-000C 24383 UGAAGGCUGGGUACCUUUG CsAsAsassUaCCCascCUUCaUsU 376 R-008356542-000T253 84 AUCUGUCAUCAAAUUGAGU B aUCUgUCaUCaaaUUgagU TsT B 377R-008356542-000T 253 84 AUCUGUCAUCAAAUUGAGU AsCsUsCaaUUUgaUgaCagaUUsU378 R-008357068-000A 254 85 UCUGUCAUCAAAUUGAGUAB UCUgUCaUCaaaUUgagUaTsT B 379 R-008357068-000A 254 85UCUGUCAUCAAAUUGAGUA UsAsCsUCaaUUUgaUgaCagaUsU 380 R-008356914-000X 25586 CUGUCAUCAAAUUGAGUAU B CUgUCaUCaaaUUgagUaU TsT B 381 R-008356914-000X255 86 CUGUCAUCAAAUUGAGUAU AsUsAsCUCaaUUUaaugaCagUsU 382R-008356733-000D 257 87 GUCAUCAAAUUGAGUAUUA B aUCaUCaaaUUgagUaUUaTsT B383 R-008356733-000D 257 87 GUCAUCAAAUUGAGUAUUAUsAsAsUaCUCaaUUUgaUgaCUsU 384 R-008356118-000D 258 88UCAUCAAAUUGAGUAUUAU B UCaUCaaaUUgagUaUUaU TsT B 385 R-008356118-000D 25888 UCAUCAAAUUGAGUAUUAU AsUsAsaUaCUCaaUUugaUgaUsU 386 R-008356115-000C279 89 UGGAGACUUCAAUUUGCCA B UagaaaCUUCaaUUUaCCaTsT B 387R-008356115-000C 279 89 UGGAGACUUCAAUUUGCCA UsGsGsCaaaUUaaaauCUCCaUsU388 R-008279398-000W 291 2 ACAACAGACUUUAAUGUAA UsUsAsCaUUaaaaUCuaUUcUUsU15 R-008279398-000W 291 2 ACAACAGACUUUAAUGUAA B aCaaCacaCUUUaaUgUaaTsT B389 R-008357241-000Z 329 90 UGGAUGAAGGCUGGGUACC B UggaUgaggCUgggUaCCTsT B 390 R-008357241-000Z 329 90 UGGAUGAAGGCUGGGUACCGsGsUsaCCCagCCUUcaUCCaUsU 391 R-008357238-000T 330 91GGAUGAAGGCUGGGUACCU B ggaUgaaggCUgggUaCCU TsT B 392 R-008357238-000T 33091 GGAUGAAGGCUGGGUACCU AsGsGsUaCCCagCCUuCaUCCUsU 393 R-008357235-000S331 92 GAUGAAGGCUGGGUACCUU B gaUgaagaCUgggUaCCUU TsT B 394R-008357235-000S 33 92 GAUGAAGGCUGGGUACCUU AsAsGsgUaCCCagCCuUCaUCUsU 395R-008357232-000R 332 93 AUGAAGGCUGGGUACCUUU B aUsaaggCUgggUaCCUUU TsT B396 R-008357232-000R 332 93 AUGAAGGCUGGGUACCUUUAsAsAsggUaCCCagCcUUCaUUsU 397 R-008357062-000Y 335 94AAGGCUGGGUACCUUUCGA B aaggCUgggUaCCUUUgaaTsT B 398 R-008357062-000Y 33594 AAGGCUGGGUACCUUUGGA UsCsCsaaasgUaCCCagCCUUUsU 399 R-008356539-000L337 95 GCCUGGGUACCUUUGGAAA B agCUaggUaCCUUUggaaaTsT B 400R-008356539-000L 337 95 GGCUGGGUACCUUUGGAAA UsUsUsCCaaagUaCcCagCCUsU 401R-008357229-000J 339 96 CUGGGUACCUUUGGAAACA B CUgggUaCCUUUggaaaCaTsT B402 R-008357229-000J 339 96 CUGGGUACCUUUGGAAACAUsGsUsUUCCaaaggUaCCCagUsU 403 R-008356908-000P 485 97GCAGACCACUCCCUGAAGU B gCagaCCaCUCCCUgaagU TsT B 404 R-008356908-000P 48597 GCAGACCACUCCCUGAAGU AsCsUsUCagggagUggUCUgCUsU 405 R-008356112-000B496 98 CCUGAAGUGACGGAUGAGU B CCUgaagUgaCggaUgagU TsT B 406R-008356112-000B 496 98 CCUGAAGUGACGGAUGAGU AsCsUsCaUCCaUCaCuUCagcUsU407 R-008279474-000L 869 99 AAAUCAUGGUGAAAUAAAAB aaaUCaUggUgaaaUaaaaTsT B 408 R-008279474-000L 869 199AAAUCAUGGUGAAAUAAAA UsUsUsUaUUUCaCCaugaUUUUsU 409 R-008357447-000W 1065100 UGCAAAUAAUGGUAACCUA B UgCaaaUaaUgaUaaCCUaTsT B 410 R-008357447-000W1065 100 UGCAAAUAAUGGUAACCUA UsAsGsgUUaCCaUUauUUgCaUsU 411R-008356730-000C 1066 101 GCAAAUAAUGGUAACCUAC B uCaaaUaaUagUaaCCUaCTsT B 412 R-008356730-000C 1066 101 GCAAAUAAUGGUAACCUACGsUsAsggUUaCCaUUaUUUgCUsU 413 R-008356727-000W 1070 102AUAAUGGUAACCUACUGUU B aUaaUggUaaCCUaCUgUU TsT B 414 R-008356727-000W1070 102 AUAAUGGUAACCUACUGUU AsAsCsagUaggUUaCcaUUaUUsU 415R-008357444-000V 1075 103 GGUAACCUACUGUUAAGGA B usUaaCCUaCUgUUaaggaTsT B416 R-008357444-000V 1075 103 GGUAACCUACUGUUAAGGAUsCsCsUUaaCaaUaggUUaCCUsU 417 R-008357226-000H 1112 104AAGUACUAGAAGGACAUGC B aagUaCUagaaggaCaUgC TsT B 418 R-008357226-000H1112 104 AAGUACUAGAAGGACAUGC GsCsAsUaUCCUUCUagUaCUUUsU 419R-008357441-000U 1304 105 AAGGAAGAGGACAGUUUCA B aaggaagaggaCacUUUCaTsT B420 R-008357441-000U 1304 105 AAGGAAGAGGACAGUUUCAUsGsAsaaCUgUCCUCuUCCUUUsU 421 R-008356109-000V 1328 106GGAGGACAAGAUUUGAUGA B ggggCaasaUUUgaUaaTsT B 422 R-008356109-000V 1328106 GGAGGACAAGAUUUGAUGA UsCsAsUCaaaUCUUsuCCUCCUsU 423 R-008356724-000V1395 107 CAGAGAAGAACCCGCAUCA B CagagaaaaaCCCgCaUCaTsT B 424R-008356724-000V 1395 107 CAGAGAAGAACCCGCAUCA UsGsAsUgCgggUUCUuCUCUgUsU425 R-008356106-000U 1397 108 GAGAAGAACCCGCAUCAAAB gagaagaaCCCgCaUCaaaTsT B 426 R-008356106-000U 1397 108GAGAAGAACCCGCAUCAAA UsUsUsgaUgCgggUUcUUCUCUsU 427 R-008384283-000V 728109 CUACACAAAUCAGCGAUUU UAGCGACUAAACACAUCAAUU 430 R-008384283-000V 728109 CUACACAAAUCAGCGAUUU UUGAUGUGUUUAGUCGCUAUU 431 R-008384280-000U 728109 CUACACAAAUCAGCGAUUU UAGCGACUAAACACAUCAAUU 430 R-008384027-000F 728109 CUACACAAAUCAGCGAUUU UUGAUGUGUUUAGUCGCUAUU 431 R-008384369-D00X 728109 CUACACAAAUCAGCGAUUU UAGCGACUAAACACAUCAAUU 432 R-008384369-000X 728109 CUACACAAAUCAGCGAUUU UUGAUGUGUUUAGUCGCUAUU 433 R-008384368-000N 728109 CUACACAAAUCAGCGAUUU UAGCGACUAAACACAUCAAUU 432 R-008384150-0000 728109 CUACACAAAUCAGCGAUUU UUGAUGUGUUUAGUCGCUAUU 433 R-008384463-000E 728109 CUACACAAAUCAGCGAUUU UAGCGACUAAACACAUCAAUU 434 R-008384463-000E 728109 CUACACAAAUCAGCGAUUU UUGAUGUGUUUAGUCGCUAUU 435 R-008384707-000P 728109 CUACACAAAUCAGCGAUUU UAGCGACUAAACACAUCAAUU 434 R-008384278-000W 728109 CUACACAAAUCAGCGAUUU UUGAUGUGUUUAGUCGCUAUU 435 R-008384549-000G 728109 CUACACAAAUCAGCGAUUU UUGAUGUGUUUAGUCGCUAUU 436 R-008384549-0000 728109 CUACACAAAUCAGCGAUUU UAGCGACUAAACACAUCAAUU 437 R-008384029-000Y 728109 CUACACAAAUCAGCGAUUU UAGCGACUAAACACAUCAAUU 437 R-008384709-000G 728109 CUACACAAAUCAGCGAUUU UUGAUGUGUUUAGUCGCUAUU 436 R-008384116-000P 728109 CUACACAAAUCAGCGAUUU UaGCGaCUaaaCaCaUCaaUU 438 R-008384116-000P 728109 CUACACAAAUCAGCGAUUU UUGaUCUGUUUaGUCGCUaUU 439 R-008384690-000A 728109 CUACACAAAUCAGCGAUUU UaGCGaCUaaaCaCaUCaaUU 438 R-008384694-000K 728109 CUACACAAAUCAGCGAUUU UUGaUCUGUUUaGUCGCUaUU 439 R-008384616-000N 728109 CUACACAAAUCAGCGAUUU UAgCgACUAAACACAUCAAUU 440 R-008384616-000N 728109 CUACACAAAUCAGCGAUUU UUgAUgUgUUUAgUCgCUAUU 441 R-008384008-000E 728109 CUACACAAAUCAGCGAUUU UAgCgACUAAACACAUCAAUU 440 R-008384119-000R 728109 CUACACAAAUCAGCGAUUU UUgAUgUgUUUAgUCgCUAUU 441 R-008384689-000L 728109 CUACACAAAUCAGCGAUUU UAGcGAcUAAAcAcAUcAAUU 442 R-008384689-000L 728109 CUACACAAAUCAGCGAUUU UUGAUGUGUUUAGUCGCUAUU 443 R-008384686-000K 728109 CUACACAAAUCAGCGAUUU UAGcGAcUAAAcAcAUcAAUU 442 R-008384006-000M 728109 CUACACAAAUCAGCGAUUU UUGAUGUGUUUAGUCGCUAUU 443 R-008383974-000K 728109 CUACACAAAUCAGCGAUUU uuGAuGuGuuuAGuCGCuAUU 444 R-008383974-000K 728109 CUACACAAAUCAGCGAUUU uAGCGACuAAACACAuCAAUU 443 R-008384447-000E 728109 CUACACAAAUCAGCGAUUU uAGCGACuAAACACAuCAAUU 445 R-008384345-000C 728109 CUACACAAAUCAGCGAUUU uuGAuGuGuuuAGuCGCuAUU 444 R-00824244f-000D 728109 CUACACAAAUCAGCGAUUU AAAUCGCUGAUUUGUGUAGUU 446 R-008242441-000D 728109 CUACACAAAUCAGCGAUUU CUACACAAAUCAGCGAUUUUU 447 R-008384722-000F 728109 CUACACAAAUCAGCGAUUU AAAUCGCUGAUUUGUGUAGUU 448 R-008384722-000F 728109 CUACACAAAUCAGCGAUUU CUACACAAAUCAGCGAUUUUU 449 R-008384297-000X 728109 CUACACAAAUCAGCGAUUU CUACACAAAUCAGCGAUUUUU 447 R-008384297-000X 728109 CUACACAAAUCAGCGAUUU AAAUCGCUGAUUUGUGUAGUU 448 R-008384558-000R 728109 CUACACAAAUCAGCGAUUU AAAUCGCUGAUUUGUGUAGUU 446 R-008384558-000R 728109 CUACACAAAUCAGCGAUUU CUACACAAAUCAGCGAUUUUU 449 R-008291632-000R 728109 CUACACAAAUCAGCGAUUU AAAUCGCUGAUUUGUGUAGUU 450 R-008291632-000R 728109 CUACACAAAUCAGCGAUUU CUACACAAAUCAGCGAUUUUU 451 R-008384383-000E 728109 CUACACAAAUCAGCGAUUU CUACACAAAUCACCGAUUUUU 447 R-008384383-000E 728109 CUACACAAAUCAGCGAUUU AAAUCGCUGAUUUGUGUAGUU 450 R-008384037-000Y 728109 CUACACAAAUCAGCGAUUU AAAUCGCUGAUUUGUGUAGUU 446 R-008384037-000Y 728109 CUACACAAAUCAGCGAUUU CUACACAAAUCAGCGAUUUUU 451 R-008384721-000X 728109 CUACACAAAUCAGCGAUUU CUACACAAAUCAGCGAUUUUU 452 R-008384721-000X 728109 CUACACAAAUCAGCGAUUU AAAUCGCUGAUUUGUGUAGUU 453 R-008384293-000M 728109 CUACACAAAUCAGCGAUUU CUACACAAAUCAGCGAUUUUU 447 R-008384293-000M 728109 CUACACAAAUCAGCGAUUU AAAUCGCUGAUUUGUGUAGUU 453 R-008384556-000Y 728109 CUACACAAAUCAGCGAUUU AAAUCGCUGAUUUGUGUAGUU 446 R-008384556-00CY 728109 CUACACAAAUCAGCGAUUU CUACACAAAUCAGCGAUUUUU 452 R-008291634-000H 728109 CUACACAAAUCAGCGAUUU AAAUCGCUGAUUUGUGUAGUU 454 R-008291634-000H 728109 CUACACAAAUCAGCGAUUU CUACACAAAUCAGCGAUUUUU 455 R-008291679-000W 728109 CUACACAAAUCAGCGAUUU CUACACAAAUCAGCGAUUUUU 447 R-008291679-000W 728109 CUACACAAAUCAGCGAUUU AAAUCGCUGAUUUGUGUAGUU 454 R-008291629-000J 728109 CUACACAAAUCAGCGAUUU AAAUCGCUGAUUUGUGUAGUU 446 R-008291629-000J 728109 CUACACAAAUCAGCGAUUU CUACACAAAUCAGCGAUUUUU 455 R-008384521-000C 728109 CUACACAAAUCAGCGAUUU aaaUCGCUGaUUUGUGUaGUU 456 R-008384521-000C 728109 CUACACAAAUCAGCGAUUU CUaCaCaaaUCaGCGaUUUUU 457 R-008384431-000K 728109 CUACACAAAUCAGCGAUUU CUACACAAAUCAGCGAUUUUU 447 R-008384431-000K 728109 CUACACAAAUCAGCGAUUU aaaUCGCUGaUUUCUGUaGUU 456 R-008384680-000H 728109 CUACACAAAUCAGCGAUUU AAAUCGCUGAUUUGUGUAGUU 446 R-008384680-000H 728109 CUACACAAAUCAGCGAUUU CUaCaCaaaUCaGCGaUUUUU 457 R-008357715-000F 728109 CUACACAAAUCAGCGAUUU AAAUCcCUgAUUUgUgUAgUU 458 R-008357715-000F 728109 CUACACAAAUCAGCGAUUU CUACACAAAUCAgCgAUUUUU 459 R-008384681-000S 728109 CUACACAAAUCAGCGAUUU CUACACAAAUCAGCGAUUUUU 447 R-008384681-000S 728109 CUACACAAAUCAGCGAUUU AAAUCgCUgAUUUgUgUAgUU 458 R-008384103-000W 728109 CUACACAAAUCAGCGAUUU AAAUCGCUGAUUUGUGUAGUU 446 R-008384103-000W 728109 CUACACAAAUCAGCGAUUU CUACACAAAUCAgCgAUUUUU 459 R-008384603-000V 728109 CUACACAAAUCAGCGAUUU cUAcAcAAAUcAGcGAUUUUU 460 R-008384603-000V 728109 CUACACAAAUCAGCAUUU AAAUcGcUGAUUUGUGUAGUU 461 R-008384602-000L 728109 CUACACAAAUCAGCGAUUU CUACACAAAUCAGCGAUUUUU 447 R-008384602-000L 728109 CUACACAAAUCAGCGAUUU AAAUCGCUGAUUUGUGUAGUU 461 R-008384234-000S 728109 CUACACAAAUCAGCGAUUU AAAUCGCUGAUUUGUGUAGUU 446 R-008384234-0008 728109 CUACACAAAUCAGCGAUUU cUAcAcAAAUcAGcGAUUUUU 460 R-008357560-000E 728109 CUACACAAAUCAGCGAUUU AAAuCGCuGAuuuGuGuAGUU 462 R-008357560-000E 728109 CUACACAAAUCAGCGAUUU CuACACAAAuCAGCGAuuuUU 463 R-008357671-000R 728109 CUACACAAAUCAGCGAUUU CUACACAAAUCAGCGAUUUUU 447 R-008357671-000R 728109 CUACACAAAUCAGCGAUUU AAAuCGCuGAuuuGuGuAGUU 462 R-008357712-000E 728109 CUACACAAAUCAGCGAUUU AAAUCGCUGAUUUGUGUAGUU 446 R-008357712-000E 728109 CUACACAAAUCAGCGAUUU CuACACAAAuCAGCGAuuuUU 463 R-008384421-000T 1016752 GUCAUCACACUCAAUACCA AUUGGUAUUCAGUGUGAUGACAC 464 R-008384421-000T10167 52 GUCAUCACACUGAAUACCA GUCAUCACACUGAAUACCAAU 465 R-008384339-000V10167 52 GUCAUCACACUGAAUACCA AUUGGUAUUCAGUGUGAUGACAC 466R-008384339-000V 10167 52 GUCAUCACACUGAAUACCA GUCAUCACACUGAAUACCAAU 467R-008384089-000C 10167 52 GUCAUCACACUGAAUACCA GUCAUCACACUGAAUACCAAU 465R-008384089-000C 10167 52 GUCAUCACACUGAAUACCA AUUGGUAUUCAGUGUGAUGACAC466 R-008384419-000V 10167 52 GUCAUCACACUGAAUACCAAUUGGUAUUCAGUGUGAUGACAC 464 R-008384419-000V 10167 52GUCAUCACACUGAAUACCA GUCAUCACACUGAAUACCAAU 467 R-008384675-000J 10167 52GUCAUCACACUGAAUACCA GUCAUCACACUGAAUACCAAU 468 R-008384675-000J 10167 52GUCAUCACACUGAAUACCA AUUGGUAUUCAGUGUGAUGACAC 469 R-008384341-000T 1016752 GUCAUCACACUGAAUACCA GUCAUCACACUGAAUACCAAU 465 R-008384341-000T 1016752 GUCAUCACACUGAAUACCA AUUGGUAUUCAGUGUGAUGACAC 469 R-008384091-000A10167 52 GUCAUCACACUGAAUACCA AUUGGUAUUCAGUGUGAUGACAC 464R-008384091-000A 10167 52 GUCAUCACACUGAAUACCA GUCAUCACACUGAAUACCAAU 468R-008384674-000A 10167 52 GUCAUCACACUGAAUACCA AUUGGUAUUCAGUGUGAUGACAC470 R-008384674-000A 10167 52 GUCAUCACACUGAAUACCA GUCAUCACACUGAAUACCAAU471 R-008384338-000L 10167 52 GUCAUCACACUGAAUACCA GUCAUCACACUGAAUACCAAU465 R-008384338-000L 10167 52 GUCAUCACACUGAAUACCAAUUGGUAUUCAGUGUGAUGACAC 470 R-008384090-000S 10167 52GUCAUCACACUGAAUACCA AUUGGUAUUCAGUGUGAUGACAC 464 R-008384090-000S 1016752 GUCAUCACACUGAAUACCA GUCAUCACACUGAAUACCAAU 471 R-008383967-000U 1016752 GUCAUCACACUGAAUACCA AUUGGUAUUCAGUGUGAUGACAC 472 R-008383967-000U10167 52 GUCAUCACACUGAAUACCA GUCAUCACACUGAAUACCAAU 473 R-008384507-000V10167 52 GUCAUCACACUGAAUACCA GUCAUCACACUGAAUACCAAU 465 R-008384507-000V10167 52 GUCAUCACACUGAAUACCA AUUGGUAUUCAGUGUGAUGACAC 472R-008384586-000A 10167 52 GUCAUCACACUCAAUACCA AUUGGUAUUCAGUGUGAUGACAC464 R-008384586-000A 10167 52 GUCAUCACACUGAAUACCA GUCAUCACACUGAAUACCAAU473 R-008039829-001W 263 110 GGACUUCUCUCAAUUUUCU ccuGAAGAGAGuuAAAAGAUU474 R-008039829-001W 263 110 GGACUUCUCUCAAUUUUCUB ucuuuuAAcucucuucAGGTT B 475 wherein: A, U, C, and G = Adenosine,Uridine, Cytidine and Guanosine ribonucleotides respectively a, u, c andg = 2′-deoxy-2′-fluoro (2′-F) modified Adenosine, Uridine, Cytidine andGuanosine respectively A, U, C and G = 2′-O-methyl (2′-OMe) modifiedAdenosine, Uridine, Cytidine and Guanosine respectively A, U, C, and G= 2′-deoxy (2′-H) modified Adenosine, Uridine, Cytidine and Guanosinerespectively B = inverted abasic T = thymidine s = phosphorothioatelinkage LB = animohexyl phosphate linker attached to an inverted abasiccap.

TABLE 2 “PC” In Vivo “PC” In Vivo “PC” in Vivo “PC” In Vivo 4hr serumApoB Efficacy Efficacy Efficacy Efficacy In Vitro Efficacy Stability(9514) (Day2) (0ay7) (Day14) (Day21) (24 hrs) (% remaining) In IC50 IC50% KD log2 R Vivo Mod % KD log2 % KD log2 % KD log2 % KD log2 (nM) (R2)(10nM) (10nM) GS PS number Stud 07/35 28 0.5 14 0.2 0.510 0.997 96 4.6 078 R-008039792- study 004D 1 07H/35U2 52 1.1 56 1.2 0.603 0.986 96 4.679 47 R-008276371- study 000S 1 Sci10 91 3.5 93 3.8 0.154 0.987 96 4.696 98 R-008277564- study 000P 1 07H/35N 10 0.2 4 0.1 −13 −0.2 −8 −0.10.489 0.998 95 4.4 19 39 R-008245595- study 000U 2 Sci10 85 2.8 95 4.392 3.6 77 2.1 0.154 0.987 96 4.6 96 98 R-008277564- study 000P 2 07H/35N16 0.3 -2 0.0 0.489 0.998 95 4.4 19 39 R-008245595- stftly 000U 3 Sci1089 3.2 95 4.4 0.154 0.987 96 4.6 96 98 R-008277684- study 000P 3 Table2: ApoB (9514) in vitro stability & potency and in vivo knockdown.Compilation of in vitro serum stability and in vivo liver mRNA knockdownvalues from polymer-conjugate delivered siRNAs. These data are depictedin Figures 11, 13A, 13B, 14A and 14B. In vitro potency was measured andis listed as an 1050 value together with the goodness-of-fit (R2) valuefrom the 1050 curve ft to the in vitro knockdown data.

TABLE 3 Vivo LNP In Vivo “LNP” In Efficacy Efficacy ApoB (9514) (Day2)(Day7) R In Vivo Mod % KD log2 % KD log2 number Study 07/35* 95 4.4 842.7 R-007887972-001 B study 4 Sci10* 92 3.6 81 2.4 R-008277560-000Estudy 4 * = siRNA does not contain C6-amino linker on 5′ of passengerstrand Table 3: ApoB (9514) in vivo knockdown. Compilation of the invivo liver mRNA knockdown values from lipid-nanoparticle deliveredsiRNAs. Note that these siRNAs differ slightly from those used in thepolymer conjugate experiments. The siRNAs in Table 3 and Figure 15B donot contain the 06-amino linker on the 5′ end of the passenger strand.

TABLE 4 “PC” In “PC” In “PC” In Efficae Efficae Efficae 4hr ApoB (Day2(Day7 (Day 14 In Vitro (24 Stabilit (% R IC50 IC50 % KD Log2 Mod % log2% 1og2 % log2 (nM) (R²) (10nM (10nM GS PS number Sci10 87 3.0 95 4.3 913.5 0.15 0.98 96 4.6 100 92 R-008277564-000P Sci10-fff 79 2.2 94 4.1 832.5 0.38 0.97 96 4.7 89 99 R-008313345-000J Sci10-ffd 88 3.1 93 3.9 883.0 0.16 0.98 96 4.5 94 100 R-008313356-000K Sci10-dfd 90 3,3 95 4.3 852.8 0.13 0.99 96 4.7 91 100 R-008313350-000H Sci10-dfin 90 3.4 92 3.6 852.8 0.13 0.99 95 4.4 92 100 R-008313344-000A Table 4: ApoB (9514) invitro stability & potency and in vivo knockdown. Compilation of the invivo mRNA knockdown and in vitro knockdown and serum stability for ApoBSci10 and related variants to the Sci10 modification motif. These dataare depicted in Figures 16 and 17. In vitro potency was measured and islisted as an IC50 value together with the goodness-of-fit (R²) valuefrom the IC50 curve-fit to the in vitro knockdown data.

TABLE 5 “PC” In Vivo “PC” In Vivo 2hr serum Efficacy Efficacy stabilitySSB (291) (Day14) (Day21) In Vitro Efficacy (24 hrs) (% remaining) 1C501C50 % KD log2 R Mod % KD log2 % KD log2 (nM) (R²) (10nM) (10nM) GS PSnumber 7H/35N 0 0.0 −5 −0.1 0.205 0.955 94 4.2 0 63 R-008245590-000ASci10 0.104 0.979 93 3.8 0 100 R -008298973-000K Sci10-dfd 0.109 0.99190 3.3 96 88 R-008313359-000L Sci10-dfm 76 2.1 63 1.5 0.089 0.976 92 3.794 99 R-008313361-000J Sci10-Ffd 78 2.2 71 1.8 0.197 0.965 94 4.0 98 99R-008308490-000W Sei10-fff 0.185 0.985 92 3.6 96 88 R-008308489-000GTable 5: SSB (291) in vitro stability & potency and in vivo knockdown.Compilation of then vivo mRNA knockdown and in knockdown and serumstability for SSB siRNAs with the 07H/35N and variants to the Sci10modification motif. These data are depicted in Figures 18, 19A, 18B, and190. In vitro potency was measured and is listed as an 1050 valuetogether with the goodness-of-fit (R2) value from the 1050 curve-fit tothe in vitro knockdown data. Scil 0, Scil Odfd, and ScilOfff were nottested in vivo and therefore no in vivo mRNA knockdown data is availablefor these siRNAs.

TABLE 6 “PC” in Vivo “PC” in Vivo “PC” in Vivo “PC” in Vivo 4hr serumEfficacy Efficacy Efficacy Efficacy in Vitro Efficacy stability ApoB(9514) (Day 2) (Day 7) (Day 14) (Pay 21) (24 hrs) (% remaining) 1C501C50 % KD log2 R Mod % KD log2 % KD log2 % KD log2 % KD log2 (nM) (R2)(10nM) (10nM) GS PS number Sci10 85 2.8 95 4.3 92 3.6 77 2.1 0.154 0.98796 4.6 96 98 R-008277564- 000P Sci11 86 2.8 88 3.1 79 2.2 38 0.7 0.0370.991 98 5.4 69 76 R-008277562- 000X Sci07f 21 0.3 21 0.3 −32 −0.4 1 0.00.293 0.992 96 4.6 100 100 R-008290704- 000W Table 6: ApoB (9514) invitro stability & potency and in vivo knockdown. These data are depictedin Figures 20A and 20B. In vitro potency was measured and is listed asan IC50 value together with the goodness-of-fit (R²) value from the IC50curve-fit to the in vitro knockdown data.

TABLE 7 “PC” In Vivo “PC” in Vivo “PC” In Vivo “PC” In Vitro 2hr serumEfficacy Efficacy Efficacy Efficacy in Vitro Efficacy stability SSB(291) (Day2) (Day7) (Day14) (Day21) (24 hrs) (% remaining) IC50 IC50 %KD log2 R Mod % KD log2 % KD log2 % KD log2 % KD log2 (nM) (R²) (10nM)(10nM) GS PS number Sci07-dfm 35 0.6 2 0.0 2 0.0 0 0.0 0.304 0.997 913.5 82 100 R-008347773- 000D Sci07-ffd 68 1.6 54 1.1 30 0.5 14 0.2 0.3220.994 92 3.7 84 100 R-008347763- 000L Sci10-dfm 84 2.6 84 2.6 76 2.1 631.5 0.089 0.976 92 3.7 94 99 R-008313361- 000J Sci10-ffd 60 1.3 70 1.778 2.2 71 1.8 0.197 0.965 94 4.0 98 99 R-008308490- 000W Table 7: SSB(291) in vitro stability & potency and in vivo knockdown. These data aredepicted in Figures 21A and 21B. In potency was measured and is listedas an 1050 value together with the goodness-of-fit (A2) value from the1050 curve-fit to the in vitro knockdown data.

TABLE 8 Non-limiting examples of Stabilization Chemistries forchemically modified siNA constructs Chemistry pyrimidine purine caps p =S Strand “Stab 00” Ribo Ribo TT at 3′-ends S/AS “Stab 1” Ribo Ribo — 5at 5′-end S/AS 1 at 3′-end “Stab 2” Ribo Ribo — All linkages Usually AS“Stab 3” 2′-fluoro Ribo — 4 at 5′-end Usually S 4 at 3′-end “Stab 4”2′-fluoro Ribo 5′ and 3′-ends — Usually S “Stab 5” 2′-fluoro Ribo — 1 at3′-end Usually AS “Stab 6” 2′-O-Methyl Ribo 5′ and 3′-ends — Usually S“Stab 7” 2′-fluoro 2′-deoxy 5′ and 3′-ends — Usually S “Stab 8”2′-fluoro 2′-O-Methyl — 1 at 3′-end S/AS “Stab 9” Ribo Ribo 5′ and3′-ends — Usually S “Stab 10” Ribo Ribo — 1 at 3′-end Usually AS “Stab11” 2′-fluoro 2′-deoxy — 1 at 3′-end Usually AS “Stab 12” 2′-fluoro LNA5′ and 3′-ends Usually S “Stab 13” 2′-fluoro LNA 1 at 3′-end Usually AS“Stab 14” 2′-fluoro 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab15” 2′-deoxy 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab 16” Ribo2′-O-Methyl 5′ and 3′-ends Usually S “Stab 17” 2′-O-Methyl 2′-O-Methyl5′ and 3′-ends Usually S “Stab 18” 2′-fluoro 2′-O-Methyl 5′ and 3′-endsUsually S “Stab 19” 2′-fluoro 2′-O-Methyl 3′-end S/AS “Stab 20”2′-fluoro 2′-deoxy 3′-end Usually AS “Stab 21” 2′-fluoro Ribo 3′-endUsually AS “Stab 22” Ribo Ribo 3′-end Usually AS “Stab 23” 2′-fluoro*2′-deoxy* 5′ and 3′-ends Usually S “Stab 24” 2′-f1uoro* 2′-O-Methyl* — 1at 3′-end S/AS “Stab 25” 2′-fluoro* 2′-O-Methyl* — 1 at 3′-end S/AS“Stab 26” 2′-f1uoro* 2′-O-Methyl* — S/AS “Stab 27” 2′-fluoro*2′-O-Methyl* 3′-end S/AS “Stab 28” 2′-fluoro* 2′-O-Methyl* 3′-end S/AS“Stab 29” 2′-fluoro* 2′-O-Methyl* 1 at 3′-end S/AS “Stab 30” 2′-f1uoro*2′-O-Methyl* S/AS “Stab 31” 2′-fluoro* 2′-O-Methyl* 3′-end S/AS “Stab32” 2′-fluoro 2′-O-Methyl* S/AS “Stab 33” 2′-fluoro 2′-deoxy* 5′ and3′-ends — Usually S “Stab 34” 2′-fluoro 2′-O-Methyl* 5′ and 3′-endsUsually S “Stab 35” 2′-fluoro*† 2′-O-Methyl*† Usually AS “Stab 36”2′-fluoro*† 2′-O-Methyl*† Usually AS “Stab04H” 2′-fluoro‡ Ribo‡ 5′ and3′-ends 1 at 3′-end Ususally S “Stab06C” 2′-O-Methyl*‡ Ribo‡ 5′ and3′-ends Ususally S “Stab07H” 2′-fluoro‡ 2′-deoxy‡ 5′ and 3′-ends 1 at3′-end Ususally S “Stab07mU” 2′-fluoro‡ 2′-deoxy‡ 5′ and 3′-endsUsusally S “Stab09H” Ribo‡ Ribo‡ 5′ and 3′-ends 1 at 3′-end Ususally S“Stab16C” Ribo‡ 2′-O-Methyl‡ 5′ and 3′-ends Ususally S “Stab16H” Ribo‡2′-O-Methyl‡ 5′ and 3′-ends 1 at 3′-end Ususally S “Stab18C” 2′-fluoro‡2′-O-Methyl‡ 5′ and 3′-ends Ususally S “Stab18H” 2 ′-fluoro‡2′-O-Methyl‡ 5′ and 3′-ends 1 at 3′-end Ususally S “Stab52H”2′-O-Methyl‡ Ribo‡ 5′ and 3′-ends 1 at 3′-end Ususally S “Stab05C”2′-fluoro‡ Ribo‡ Ususally AS “Stab05N” 2′-fluoro‡ Ribo‡ 1 at 3′-endUsusally AS “Stab10C” Ribo‡ Ribo‡ Ususally AS “Stab10N” Ribo‡ Ribo‡ 1 at3′-end Ususally AS “Stab35G*” 2′-fluoro‡ 2′-O-Methyl‡ Ususally AS“Stab35N*” 2′-fluoro‡ 2′-O-Methyl‡ 1 at 3′-end Ususally AS “Stab35rev*”2′-O-Methyl‡ 2′-fluoro‡ Ususally AS “Stab50*” Ribo‡ 2′-O-Methyl‡Ususally AS “Stab53*” 2′-O-Methyl‡ Ribo‡ Ususally AS “Stab53N*”2′-O-Methyl‡ Ribo‡ 1 at 3′-end Ususally AS Stab54 Ribo‡ 2′-fluoro‡Ususally AS CAP = any terminal cap, see for example FIGS. 6 and 10. AllStab chemistries can be used in combination with each other for duplexesof the invention (e.g., as combinations of sense and antisense strandchemistries), or alternately can be used in isolation, e.g., for singlestranded nucleic acid molecules of the invention. All Stab chemistriescan comprise 3′-overhang nucleotides having 2′-O-alkyl,2′-deoxy-2′-fluoro, 2′-deoxy, LNA or other modified nucleotides ornon-nucleotides. All Stab chemistries typically comprise about 19-21nucleotides, but can vary as described herein. All Stab chemistries canalso include a single ribonucleotide in the sense or passenger strand atthe 11^(th) base paired position of the double-stranded nucleic acidduplex as determined from the 5′-end of the antisense or guide strand(see FIG. 5C). All Stab chemistries can also have a 2′-deoxy-2′-fluoromodification at position 14 from the 5′ end of the antisense strandregardless of whether it is a purine or pyrimidine at that position (seeFIGS. 5C and 12). All Stab chemistries of the antisense strand presentedabove can have a thymidine in place of a 2′-deoxy uridine at position 1,2, and/or 3 from the 5′ end of the antisense strand (see FIG. 5C). AllStab chemistries can include a plurality of the specified purine and/orpyrimidine chemistries. S = sense strand. AS = antisense strand *Stab 23has a single ribonucleotide adjacent to 3′-CAP. *Stab 24 and Stab 28have a single ribonucleotide at 5′-terminus. *Stab 25, Stab 26, Stab 27,Stab 35, Stab 35G*, Stab 35N*, Stab35rev*, Stab 36, Stab 50*, Stab53*,Stab 53N*, and Stab 54 have three ribonucleotides at 5′-terminus. *Stab29, Stab 30, Stab 31, Stab 33, and Stab 34 any purine at first threenucleotide positions from 5′-terminus are ribonucleotides. p =phosphorothioate linkage. †Stab 35 has 2′-O-methyl U at 3′-overhangs andthree ribonucleotides at 5′-terminus. †Stab 36 has 2′-O-methyl overhangsthat are complementary to the target sequence. (naturally occurringoverhangs) and three ribonucleotides at 5′-terminus. ‡Stab 04H, Stab06C, Stab07H, Stab07mU, Stab09H, Stab16C, Stab16H, Stab18C, Stab18H,Stab 52H, Stab05C, Stab05N, Stab10C, Stab10N, Stab35G*, Stab35N*,Stab35N*, Stab35rev*, Stab50*, Stab53*, Stab 53N*, Stab54 have two2′-O-methyl U 3′-overhangs. Stab35G*, Stab 35N*, Stab35rev*, Stab50*,Stab53*, and Stab53N* do not allow for a 2′-O-methyl modification atposition 14 of the guide strand as determined from the 5′-end.

TABLE 9 A. 2.5 μmol Synthesis Cycle ABI 394 Instrument Wait Time* WaitTime* Wait Reagent Equivalents Amount DNA 2-O-methyl Time*RNAPhosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-Ethyl 23.8 238 μL45 sec 2.5 min 7.5 min Tetrazole Acetic 100 233 μL 5 sec 5 sec 5 secAnhydride N-Methyl 186 233 μL 5 sec 5 sec 5 sec Imidazole TCA 176 2.3 mL21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage12.9 645 μL 100 sec 300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B.0.2 μmol Synthesis Cycle ABI 394 Instrument Wait Time* Wait Time* WaitReagent Equivalents Amount DNA 2-O-methyl Time*RNA Phosphoramidites 1531 μL 45 sec 233 sec 465 sec S-Ethyl 38.7 31 μL 45 sec 233 min 465 secTetrazole Acetic 655 124 μL 5 sec 5 sec 5 sec Anhydride N-Methyl 1245124 μL 5 sec 5 sec 5 sec Imidazole TCA 700 732 μL 10 sec 10 sec 10 secIodine 20.6 244 μL 15 sec 15 sec 15 sec Beaucage 7.7 232 μL 100 sec 300sec 300 sec Acetonitrile NA 2.64 mL NA NA NA C. 0.2 μmol Synthesis Cycle96 well Instrument Equivalents Amount DNA/2′-O- DNA/2′-O- Wait Time*Wait Time* Wait Time* Reagent methyl/Ribo methyl/Ribo DNA 2-O-methylRibo Phosphoramidites 22/33/66 40/60/120 μL 60 sec 180 sec 360 secS-Ethyl 70/105/210 40/60/120 μL 60 sec 180 min 360 sec Tetrazole Acetic265/265/265 50/50/50 μL 10 sec 10 sec 10 sec Anhydride N-Methyl502/502/502 50/50/50 μL 10 sec 10 sec 10 sec imidazole TCA 238/475/475250/500/500 μL 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 μL 30sec 30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 700 sec 200 secAcetonitrile NA 1150/1150/1150 μL NA NA NA Wait time does not includecontact time during delivery. Tandem synthesis utilizes double couplingof linker molecule

TABLE 10 09H/1 ON Sci10 10nm 1nm diff target l0nM 1nM l0nM 1nM diff fromfrom target site R# KD 10nM KD KD KD 10nM KD 1nM 09H/10N 09H/10N (iog2)(log2) KD (%) (log2) (log2) R# (log2) KD (%) (log2) KD (%) (log2) (log2)APOB 19 R-008357258-000C 4.97 97 4.08 94 R-008355979- 1.58 67 0.55 32−3.39 −3.53 000A APOB 248 R-008357080-000R 5.63 98 4.27 95 R-008356396-4.51 96 3.37 90 −1.12 −0.90 000V APOB 397 R-008355914-0000 4.05 94 2.5983 R-008357291- 0.74 40 0.23 15 −3.31 −2.36 000L APOB 485R-008356933-000Y 5.97 98 4.76 96 R-008357122- 5.47 98 4.80 96 −0.50 0.05000N APOB 601 R-008356751-000W 4.15 94 3.49 91 R-008355976- 2,91 87 1.1354 −1.24 −2.36 000Z APOB 719 R-008355911-000B 5.64 98 4.13 94R-008357288- 1.68 69 1.36 61 −3.96 −2.77 000E APOB 780 R-008356343-000G4.29 95 2.79 86 R-008356569- 0.05 3 −0.06 −4 −4.24 −2.84 000N APOB 1124R-008357252-000A 4.49 96 3.63 92 R-008356393- 5.33 98 4.44 95 0.84 0.80000U APOB 1445 R-008356340-000F 5.18 97 3.51 91 R-008355973- 2.07 760.79 42 −3.10 −2.73 000Y APOB 1446 R-008357255-000B 5.72 98 4.34 95R-.008356941- 5.40 98 3.97 94 −0.32 −0.37 000Y APOB 1983R-008356337-000Z 5.17 97 4.05 94 R-008356184- 5.29 97 4.59 96 0.11 0.54000R APOB 3214 R-008355917-000D 3.68 92 2.33 80 R-.008356351- 116 891.39 62 −0.52 0.94 000G APOB 3614 R-008357077-000J 4.25 95 4.08 94R-008356795- 4.69 96 4.17 94 0.09 0.43 000A APOB 4542 R-008356128-000W4.08 94 3.30 90 R-008356604- 5.07 97 4.54 96 1.24 0.99 000A APOB 6548R-008356561-000U 5.17 97 4.36 95 R-008356134- 5.06 97 3.75 93 0.11 −0.61000D APOB 6930 R-008355905-000U 2.83 86 1.38 61 R-008357119- 3.23 891.66 68 0.39 0.28 000G APOB 6981 R-008356558-000M 4.54 96 3.59 92R-008356181- 5.22 97 4.41 95 0.67 0.82 000P APOB 7044 R-008357083-000S5.60 98 4.69 96 R-008355923- 4.76 96 3.66 92 −0 84 −1.03 000L APOB 9414R-008356334-000Y 5.21 97 4.78 96 R-008356969- 4.56 96 3.81 93 −0.64−0.96 000C APOB 9514 R-008357249-000U 4.95 97 3.41 91 R-008277560- 4.7096 3.92 93 −0.24 0.51 000E APOB 9621 R-008356555-000L 3.47 91 2.73 85R-008356767- 4.17 94 2.37 81 0.70 −0.36 000R APOB 10162 R-008356930-000X3.61 92 3.67 92 R-008356601- 2.57 83 1.11 54 −1.04 −2.56 000Z APOB 10167R-008356552-000K 3.62 92 3.70 92 R-008356598- 3.30 90 1.74 70 −0.31−1.97 000G APOB 10168 R-008356331-000X 4.48 96 4.39 95 R-008279809- 2.5383 1.48 64 −1.95 −2.91 000X APOB 10219 R-008356125-000V 2.91 87 1.51 65R-008355970- 2.25 79 1.03 51 −0.66 −0.48 000X APOB 10455R-008356549-000D 4.50 96 4.09 94 R-008355967- 4.46 95 2.91 87 −0.04−1.18 000R APOB 10517 R-008356329-000Z 4.34 95 3.15 89 R-008356178- 4.7596 3.33 90 0.41 0.18 000H APOB 12673 R-008356326-000Y 4.64 96 4.22 95R-008356792- 3.60 92 3.14 89 −1.04 −1.08 000Z APOB 13666R-008356748-000P 4.90 97 4.44 95 R-008356387- 5.09 97 4.68 96 0.19 0.24000L Table 10: Sci10 tolerance measured in 29 ApoB siRNAs. Knockdown oftarget mRNA was measured at l0nM and 1nM concentrations to estimate theimpact of Scil0 modification on the potency of the tested siRNAs.Comparison of 09H/10N and Scil0 was performed on a pair-wise basis foreach of the 29 different siRNA sequences. The difference in knockdown(in log 2) was calculated by subtracting 09H/10N knockdown levels fromthose measured for the Scil0 modification pattern. Positive valuesindicate the Scil0 modification pattern is more active than minimallymodified 09H/10N; overall a rare event for modified siRNAs. Negativevalues indicate that the Scil0 modification is less active relative to09H/10N The experimental variation and accuracy of the qPCR assay isapproximately 0.5 (1og2). Values within 0.5 of the 09H/10N areconsidered to be equivalent in overall knockdown and therefore equallytolerated, Overall, 15 (52%) ApoB siRNAs tolerate the Scil0 motif atlOnM and 13 (45%) at lnM siRNA concentration.

TABLE11 09H/10N Sci10 10nm 1nm diff l0nM 1nM 10nM 1nM diff from fromtarget KD 10nM KD 1nM KD 10nM KD 1nM 09H/10N 09H/10N target site R#(log2) KD (%) (log2) KD (%) R# (log2) KD (%) (log2) KD (%) (log2) (log2)PHD2 70 R-008391240- 4.28 95 3.10 88 R-008391351- 2.99 87 1.61 67 −1.29−1.49 000E 000R PHD2 93 R-008391213- 4.83 96 3.05 88 R-008391293- 4.9797 3.06 88 0.14 0.02 000D 000T PHD2 146 R-008313809- 4.81 96 3.13 89R-008391258- 4.71 96 3.82 93 −0.10 0.69 000Y 000S PHD2 196 R-008313864-4.60 96 2.77 85 R-008391290- 4.79 96 3.47 91 0.19 0.70 000J 000S PHD2284 R-008391328- 4.05 94 2.98 87 R-008391372- 3.72 92 1.97 74 −0.33−1.01 000Z 000J PHD2 384 R-008391263- 4.07 94 3.03 88 R-008391348- 3.9193 2.92 87 −0.17 −0.11 000R 000J PHD2 420 R-008391207- 3.53 91 2.06 76R-008391287- 0.40 24 0.00 0 −3.13 −2.05 000W 000K PHD2 485 R-008391296-2.06 76 0.75 41 R-008391345- 0.24 16 0.03 2 −1.81 −0.72 000U 000H PHD2661 R-008391228- 3.83 93 2.28 79 R-008391311- 3.89 93 2.35 80 0.06 0.07000P 000W PHD2 780 R-008391414- 3.60 92 2.22 79 R-008391369- 0.94 480.11 7 −2.66 −2.12 000G 000C PHD2 849 R-008391411- 3.76 93 2.47 82R-008391342- 1.18 56 0.26 17 −2.58 −2.21 000F 000G PHD2 881 R-008391314-2.58 83 0.73 40 R-008391366- 3.54 91 1.35 61 0.96 0.62 000X 000B PHD2887 R-008391325- 3.36 90 1.74 70 R-008391405- 4.21 95 2.10 77 0.85 0.37000Y 000Y PHD2 955 R-008350794- 2.14 77 0.44 26 R-008391255- 4.17 942.27 79 2.03 1.83 000Z 000R PHD2 962 R-008350713- 3.23 89 1.11 54R-008391402- 3.42 91 1.69 69 0.19 0.58 000B 000X PHD2 994 R-008391266-2.92 87 1.16 55 R-008391192- 0.95 48 0.21 13 −1.97 −0.95 000S 000Z PHD21048 R-008391357- 3.89 93 2.16 78 R-008391284- 0.55 32 0.04 3 −3.34−2.12 000T 000J PHD2 1055 R-008391234- 4.94 97 3.25 89 R-008391281- 5.1197 3.65 92 0.17 0.40 000X 000H PHD2 1107 R-008391302- 3.35 90 2.29 80R-008391201- 0.48 28 0.09 6 −2.88 −2.20 000M 000U PHD2 1115 R-008391299-2.19 78 0.73 40 R-008391252- 3.06 88 1.12 54 0.87 0.39 000V 000P PHD21223 R-008391354- 3.56 92 2.33 80 R-008391198- 3.84 93 2.50 82 0.28 0.16000S 000B PHD2 4295 R-008313818- 3.74 93 2.62 84 R-008391249- 2.13 771.28 59 −1.61 −1.33 000G 000H PHD2 4302 R-008313815- 3.55 91 2.96 87R-008391246- 2.36 80 1.36 61 −1.19 −1.60 000F 000G PHD2 4381R-008391381- 3.62 92 2.24 79 R-008391222- 3.41 91 1.95 74 −0.20 −0.29000T 000M Table 11: Sci10 tolerance measured in 24 PI-11)2 siRNAs.Knockdown of target mRNA was measured at 10nM and 1nM concentrations toestimate the impact of Sci10 modification on the potency of the testedsiRNAs Comparison of 09H/ION and Scil0 was performed on a pair-wisebasis for each of the 24 different siRNA sequences. The difference inknockdown (in log 2) was calculated by subtracting 091-I/10N knockdownlevels from those measured for the Sci10 modification pattern. Positivevalues indicate the Sci10 modification pattern is more active thanminimally modified 09H/ 10N; overall a rare event for modified siRNAs.Negative values indicate that the Scil0 modification is less activerelative to 09H/10N. The experimental variation and accuracy of the qPCRassay is approximately 0.5 (10g2). Values within 0.5 of the 0911110N areconsidered to be equivalent in overall knockdown and therefore equallytolerated. Overall, 14 (58%) PHD2 siRNAs tolerate the Scil0 motif atlOnM and 13 (54%) at lriM siRNA concentration.

TABLE 12 09H/10N Sci10 10nm 1nm diff 10nM 1nM 10nM 1nM diff from fromtarget KD 10nM KD 1nM KD 10nM KD 1nM 09H/10N 09H/10N target site R#(log2) KD (%) (log2) KD (%) R# (log2) KD (%) (log2) KD (%) (log2) (log2)SSB 243 R-008357193- 2.21 78 1.19 56 R-008357450- 0.11 7 0.15 10 −2.10−1.04 000U 000C SSB 253 R-008356271- 2.92 87 2.61 84 R-008356542- 2.2479 1.45 63 −0.69 −1.16 000G 000T SSB 254 R-008356480- 2.80 86 2.38 81R-008357068- 3.04 88 2.03 76 0.24 −0.35 000K 000A SSB 255 R-008356688-3.72 92 3.14 89 R-008356914- 3.03 88 2.92 87 −0.69 −0.21 000Z 000X SSB257 R-008357396- 3.40 91 2.87 86 R-008356733- 4.23 95 3.91 93 0.84 1.04000P 000D SSB 258 R-008356265- 3.85 93 3.65 92 R-008356118- 3.96 94 3.9393 0.12 0.28 000Z 000D SSB 279 R-008357199- 2.50 82 1.78 71 R-008356115-1.29 59 0.82 43 −1.21 −0.96 000W 000C SSB 291 R-008356273- 4.00 94 4.0094 R-008279398- 4.66 96 3.99 94 0.66 −0.01 000Z 000W SSB 329R-008356262- 2.53 83 1.89 73 R-008357241- 0.05 3 0.07 5 −2.48 −1.82 000Y000Z SSB 330 R-008357393- 2.89 87 2.76 85 R-008357238- 2.08 76 1.30 59−0.81 −1.46 000N 000T SSB 331 R-008357040- 3.38 90 3.53 91 R-008357235-3.00 88 2.44 82 −0.38 −1.09 000W 000S SSB 332 R-008356477- 3.78 93 3.4291 R-008357232- 0.64 36 0.55 32 −3.14 −2.87 000D 000R SSB 335R-008356871- 2.81 86 2.61 84 R-008357062- 3.34 90 2.84 86 0.53 0.23 000R000Y SSB 337 R-008357390- 2.72 85 2.03 76 R-008356539- 2.13 77 1.57 66−0.60 −0.46 000M 000L SSB 339 R-008356060- 3.67 92 3.20 89 R-008357229-0.16 11 0.02 1 −3.51 −3.18 000L 000J SSB 485 R-008357196- 3.45 91 2.9587 R-008356908- 0.15 10 0.05 3 −3.30 −2.91 000V 000P SSB 496R-008356057- 3.16 89 2.72 85 R-008356112- 1.84 72 1.20 57 −1.33 −1.52000E 000B SSB 869 R-008356275- 3.81 93 3.90 93 R-008279474- 3.52 91 3.6792 −0.30 −0.24 000S 000L SSB 1065 R-008357190- 4.08 94 4.03 94R-008357447- 4.07 94 3.74 92 −0.01 −0.29 000T 000W SSB 1066 R-008357037-3.84 93 3.44 91 R-008356730- 2.78 85 2.54 83 −1.06 −0.90 000P 000C SSB1070 R-008356483- 3.68 92 3.59 92 R-008356727- 3.65 92 3.47 91 −0.03−0.12 000L 000W SSB 1075 R-008356259- 2.56 83 1.97 74 R-008357444- 1.0351 0.47 28 −1.53 −1.50 000S 000V SSB 1112 R-008356682- 2.27 79 1.65 68R-008357226- 0.10 7 0.17 11 −2.18 −1.48 000X 000H SSB 1304 R-008356278-3.86 93 3.50 91 R-008357441- 3.03 88 2.85 86 −0.83 −0.65 000T 000U SSB1328 R-008356054- 3.19 89 2.78 85 R-008356109- 2.18 78 1.94 74 −1.01−0.84 000D 000V SSB 1395 R-008356471- 2.87 86 2.63 84 R-008356724- 1.3962 1.32 60 −1.47 −1.31 000B 000V SSB 1397 R-008357387- 3.79 93 3.89 93R-008356106- 3.65 92 3.57 92 −0.14 −0.32 000F 000U Table 12: Sci10tolerance measured in 27 SSB siRNAs. Knockdown of target mRNA wasmeasured at 10nM and lnM concentrations to estimate the impact of Scil0modification on the potency of the tested siRNAs. Comparison of 09H/10Nand Sci10 was performed on a pair-wise basis for each of the 27different siRNA sequences. The difference in knockdown (in log 2) wascalculated by subtracting 09H/10N knockdown levels from those measuredfor the Sci10 modification pattern. Positive values indicate the Sci10modification pattern is more active than minimally modified 09H/10N;overall a rare event for modified siRNAs. Negative values indicate thatthe Scii0 modification is less active relative to 09H/10N. Theexperimental variation and accuracy of the qPCR assay is approximately0.5 (1og2). Values within 0.5 of the 09H/10N are considered to beequivalent in overall knockdown and therefore equally tolerated.Overall, 10 (37%) SSB siRNAs tolerate the Scil0 motif at l0nM and 11(41%) at 1nM siRNA concentration.

TABLE 13 Table 13: Summary of data from Tables 10-12. 1og2 differencefrom unmodified (10 nM) modification pattern <−0.25 <−0.50 <−0.75 <−1.00Sci10 33    39    45    48    (41%) (49%) (56%) (60%) 1og2 differencefrom unmodified (1 nM) modification pattern <−0.25 <−0.50 <−0.75 <−1.00Sci10 29    37    40    47    (36%) (46%) (50%) 09%) Knockdown of targetmRNA was measured at 10 nM and 1 nM concentrations to estimate theimpact of Sci10 modification on the potency of the tested siRNAs.Comparison of 09H/10N and Sci10 was performed on a pair-wise basis foreach of the 80 different siRNA sequences shown in Tables 10-12. Thedifference in knockdown (in log 2) was calculated by subtracting 09H/10Nknockdown levels from those measured for the Sci10 motif. Positivevalues indicate the Sci10 motif is more active than minimally modified09H/10N; overall a rare event for modified siRNAs. Negative valuesindicate that the Sci10 modification is deleterious relative to 09H/10N.The experimental variation and accuracy of the qPCR assay isapproximately 0.5 (log2). Values within 0.5 of the 09H/10N areconsidered to be equivalent in overall knockdown and therefore equallytolerated.

TABLE 14 fold-reduction of TNFa number of (relative to unmoded)modifications GS & PS GS PS GS & PS GS PS 2′OMe 2′F 2′OMe 2′F 2′OMe 2′F# % # % # % B-gal control sRNA cytidine — — — — — — 7 17 5 12 2 5uridine 77 6 2 — 7 4 12 29 3 7 9 21 guanosine 42 2 17 — 3 2 7 17 2 5 512 adenosine 108 142 133 51 110 39 12 29 9 21 3 7 B-gal 728 siRNAcytidine 2 — — — — — 7 17 2 5 5 12 undine 52 4 51 — 33 — 12 29 7 17 5 12guanosine 47 18 — 15 — — 7 17 5 12 2 5 adenosine 49 13 50 4 47 7 12 29 512 7 17 Table 14: Fold-reduction of TNF-alpha levels for 2 ribosemodifications relative to unmodified RNA. Dashes indicate values where2' modification had less than two-fold reduction of siRNA mediated TNFainduction (e.g. cytidine). The number of modifications per oligo aretotaled and listed as a percentage of the overall oligo (counting bothpassenger and guide strands).

TABLE 15 Table 15: TNFa TNFa Beta-gal Beta-gal AVG SD activity (%activity siRNA R-number (ng/mL) (ng/mL) activity) (SD) Bgal-control 2′OHunmod R-008384290-000L 3.78 1.31 85.2 9.6 Bgal-cntrol 2′OMe A-GS &R-008384283-000V 0.04 0.01 88.6 23.3 PS Bgal-control 2′OMe A-GSR-003384280-000U 0.03 0.01 92.8 8.6 Bgal-control 2′OMe A-PSR-008384027-000F 0.03 0.02 99.9 9.6 Bgal-control 2′OMe G-GS &R-008384369-000X 0.09 0.05 91.9 11.6 PS Bgal-control 2′OMe G-GSR-008384368-000N 0.23 0.18 91.3 13.4 Bgal-control 2′OMe G-PSR-008384150-000G 1.16 0.88 92.1 4.4 Bgal-control 2′OMe C-GS &R-008384463-000E 3.62 0.97 96.7 5.7 PS Bgal-control 2′OMe C-GSR-003384707-000P 3.97 1.56 99.4 10.1 Bgal-control 2′OMe C-PSR-008384278-000W 3.33 0.86 91.9 5.5 Bgal-control 2′OMe U-GS &R-008384549-000G 0.05 0.02 103.6 7.9 PS Bgal-control 2′OMe U-GSR-008384029-000Y 1.53 0.90 80.4 11.8 Bgal-control 2′OMe U-PSR-008384709-000G 0.55 0.55 91.4 14.9 Bgal-control 2′F A-GS &R-008384116-000P 0.03 0.02 88.9 12.0 PS Bgal-control 2′F A-GSR-003384690-000A 0.07 0.08 90.7 13.9 Bgal-control 2′F A-PSR-008384694-000K 0.10 0.05 98.1 14.5 Bgal-control 2′F C-GS &R-008384616-000N 1.74 0.52 89.6 11.1 PS Bgal-control 2′F C-GSR-008384008-000E 3.97 0.66 91.0 15.4 Bgal-control 2′F G-PSR-008384119-000R 1.73 0.13 88.2 10.2 Bgal-control 2′F C-GS &R-008384689-000L 3.11 1.11 96.4 5.3 PS Bgal-control 2′F C-GSR-008384686-000K 4.10 0.66 90.5 7.5 Bgal-control 2′F C-PSR-003384006-000M 5.05 0.27 93.3 12.4 Bgal-control 2′F U-GS &R-008383974-000K 0.65 0.36 94.3 9.7 PS Bgal-control 2′F U-GSR-008384447-000E 4.27 0.59 82.7 17.3 Bgal-control 2′F U-PSR-008384345-000C 1.02 0.41 81.0 15.3 TNF-alpha levels (nanograms per ML)measured from in vitro human PBMC assay and Beta-galactosidase enzymeactivity (%) measured from the cell-based CMV-Sport B-gal transgeneassay. Values for B-gal control siRNA are shown.

TABLE 16 Table 16: TNFa TNFa Beta-gal Beta-gal AVG SD activity (%activity siRNA R-number (ng/ml) (ng/ml) activity) (SD) Bgal-728 2′OHunmod R-008242441-000D 1.50 0.86 17.8 5.6 Bgal-728 2′OMe A-GS & PSR-008384722-000F 0.03 0.02 89.5 13.3 Bgal-728 2′OMe A-GSR-008384297-000X 0.03 0.02 60.5 20.4 Bgal-728 2′OMe A-PSR-008384558-000R 0.03 0.02 17.4 6.3 Bgal-728 2′OMe G-GS & PSR-008291632-000R 0.03 0.02 64.4 10.2 Bgal-728 2′OMe G-GSR-008384383-000E 0.08 0.10 65.8 16.1 Bgal-728 2′OMe G-PSR-008384037-000Y 0.10 0.09 18.4 6.1 Bgal-728 2′OMe C-GS & PSR-008384721-000X 0.99 0.46 26.7 11.6 Bgal-728 2′OMe C-GSR-008384293-000M 1.30 0.75 25.6 7.2 Bgal-728 2′OMe C-PS R-008384556-000Y1.40 0.71 18.1 5.4 Bgal-728 2′OMe U-GS & PS R-008291634-000H 0.03 0.0220.5 7.0 Bgal-728 2′OMe U-GS R-008291679-000W 0.03 0.02 20.2 5.7Bgal-728 2′OMe U-PS R-008291629-000J 0.05 0.03 17.2 5.8 Bgal-728 2′FA-GS & PS R-008384521-000C 0.11 0.00 21.9 6.5 Bgal-728 2′F A-GSR-008384431-000K 0.34 0.02 19.8 7.4 Bgal-728 2′F A-PS R-008384680-000H0.23 0.03 21.1 7.4 Bgal-728 2′F G-GS & PS R-008357715-000F 2.38 0.7113.2 3.4 Bgal-728 2′F G-GS R-008384681-000S 2.62 0.56 12.7 3.3 Bgal-7282′F G-PS R-008384103-000W 2.87 0.66 17.9 5.3 Bgal-728 2′F C-GS & PSR-008384603-000V 1.62 0.15 17.9 5.5 Bgal-728 2′F C-GS R-008384602-000L2.31 0.25 17.5 4.8 Bgal-728 2′F C-PS R-008384234-000S 3.15 0.94 17.7 3.7Bgal-728 2′F U-GS & PS R-008357560-000E 0.39 0.03 31.2 12.2 Bgal-728 2′FU-GS R-008357671-000R 2.19 0.32 26.2 7.9 Bgal-728 2′F U-PSR-008357712-000E 2.12 0.27 18.4 6.0 TNF-alpha levels (nanograms per mL)measured from in vitro human PBMC assay and Beta-galactosidase enzymeactivity (%) measured from the cell-based CMV-Sport B-gal transgeneassay. Values for B-gal 728 siRNA are shown.

TABLE 17 Table 17: TNFa TNFa AVG SD siRNA R-number (ng/ml) (ng/ml) ApoBunmod R-008384421-000T 3.93 0.66 ApoB 2′OMe A-GS & PS R-008384339-000V0.05 0.01 ApoB 2′OMe A-GS R-008384089-000C 0.06 0.01 ApoB 2′OMe A-PSR-008384419-000V 0.06 0.01 ApoB 2′OMe G-GS & PS R-008384675-000J 0.060.01 ApoB 2′OMe G-GS R-008384341-000T 0.06 0.01 ApoB 2′OMe G-PSR-008384091-000A 0.24 0.08 ApoB 2′OMe C-GS & PS R-008384674-000A 2.210.90 ApoB 2′OMe C-GS R-008384338-000L 2.25 0.37 ApoB 2′OMe C-PSR-008384090-000S 2.42 0.68 ApoB 2′OMe U-GS & PS R-008383967-000U 0.050.01 ApoB 2′OMe U-GS R-008384507-000V 0.06 0.01 ApoB 2′OMe U-PSR-008384586-000A 0.19 0.05 TNF-alpha levels (nanograms per mL) measuredfrom in vitro human PBMC assay. Values for ApoB siRNA are shown.

TABLE 18 Composition of certain Lipid Nanoparticle Formulations siNALipid Components and Molar Ratios Duplex NTP Compound Cholesterol DSPCPEG-DMG Any siNA duplex 6 32 (30%) (10%) (2%) of the invention (50%) N/Pratio = Nitrogen:Phosphorous ratio between cationic lipid and nucleicacid.

TABLE 19 Lipid Chemical Structure Compound 32 Cholesterol DSPC PEG-DMG

Chemical Structures of Lipids in Formulations of Table 18

What we claim is:
 1. A composition comprising: I) a double-strandedshort interfering nucleic acid (siNA) molecule that inhibits theexpression of a target gene via RNA interference, having a sense strandand an antisense strand and comprising formula (A):

wherein, the upper strand is the sense strand and the lower strand isthe antisense strand of the double-stranded nucleic acid molecule;wherein the antisense strand comprises a sequence having at least 15nucleotides that are complementary to a target RNA sequence encoded bythe target gene and the sense strand comprises a sequence that iscomplementarity to the antisense strand; each N is independently anucleotide which is unmodified or chemically modified or is optionally anon-nucleotide; each B is independently a terminal cap that is presentor absent; (N) represents overhanging nucleotides, each of which isindependently unmodified or chemically modified; [N] representsnucleotides at the 5′-terminus of the antisense strand; X1 and X2 areindependently integers from 0 to 4; X3 is an integer from 15 to 30; X4is an integer from 12 to 27; and X5 is an integer from 1-6, providedthat the sum of X4 and X5 is an integer from 15-30; and wherein (a) fiveor more pyrimidine nucleotides in N_(X4) positions are 2′-O-alkylnucleotides; (b) five or more purine nucleotides in N_(X4) positions are2′-halo nucleotides; (c) five or more pyrimidine nucleotides in N_(X3)positions are 2′-O-alkyl nucleotides; (d) five or more purinenucleotides in N_(x3) positions are 2′-halo nucleotides; and (e) [N]position nucleotide(s) are any combination of ribonucleotides,deoxyribonucleotides, 2′-O-alkyl nucleotides, or 2′-halo nucleotides;and II) a cationic lipid comprising13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13 16-dien-1-amine.
 2. Thecomposition according to claim 1, wherein: (a) five or more pyrimidinenucleotides in N_(X4) positions are 2′-O-methyl nucleotides; (b) five ormore purine nucleotides in N_(X4) positions are 2′-deoxy-2′-fluoronucleotides; (c) five or more pyrimidine nucleotides in N_(X3)positionsare 2′-O-methyl nucleotides; (d) five or more purine nucleotides inN_(X3) positions are 2′-deoxy-2′-fluoro nucleotides; and (e) [N]position nucleotide(s) are any combination of ribonucleotides,deoxyribonucleotides, 2′-O-alkyl nucleotides, or 2′-halo nucleotides. 3.The composition according to claim 1, wherein: (a) 5, 6, 7, 8, 9, 10 ormore pyrimidine nucleotides in N_(X4) positions are 2′-O-methylnucleotides; (b) 5, 6, 7, 8, 9, 10 or more purine nucleotides in N_(X4)positions are 2′-deoxy-2′-fluoro nucleotides; (c) 5, 6, 7, 8, 9, 10 ormore pyrimidine nucleotides in N_(X3) positions are 2′-O-methylnucleotides; (d) 5, 6, 7, 8, 9, 10 or more purine nucleotides in N_(X3)positions are 2′-deoxy-2′-fluoro nucleotides; and (e) [N] positionnucleotide(s) are any combination of ribonucleotides,deoxyribonucleotides, 2′-O-alkyl nucleotides, or 2′-halo nucleotides. 4.The composition according to claim 1, wherein X5 is
 3. 5. Thecomposition according to claim 4, wherein the three [N] nucleotides offormula (A) are represented as 5′-[N1, N2, N3]-3′, wherein: a. each N1,N2, and N3 is a ribonucletide; or b. each N1, N2, and N3 is a2′-deoxy-2′-fluoro nucleotide; or c. each N1, N2, and N3 is a 2′-deoxynucleotide; or d. each N1, N2, and N3 is a 2′-O-alkyl nucleotide; and e.any of N1, N2, or N3 optionally comprises a phosphorothioateinternucleotide linkage.
 6. The composition according to claim 4,wherein the three [N] nucleotides of formula (A) are represented as5′-[N1, N2, N3]-3′, wherein: a. N1 is a 2′-deoxy-2′-fluoro nucleotide,N2 is 2′-deoxy-2′-fluoro nucleotide, and N3 is a 2′-deoxynucleotide; andb. any of N1, N2, or N3 optionally comprises a phosphorothioateinteraucleotide linkage.
 7. The composition according to claim 4,wherein the three [N] nucleotides of formula (A) are represented as5′-[N1, N2, N3]-3′, wherein: a. N1 is a 2′-deoxy nucleotide, N2 is2′-deoxy-2′-fluoro nucleotide, and N3 is a 2-O-methyl nucleotide; and b.any of N1, N2, or N3 optionally comprises a phosphorothioateinternucleotide linkage.
 8. The composition according to claim 4,wherein the three [N] nucleotides of formula A) are represented as5″-[N1, N2, N3]-3′, wherein: a. N1 is a 2′-deoxy nucleotide, N2 is2′-deoxy-2′-fluoro nucleotide, and N3 is a 2′-deoxy nucleotide; and b.any of N1, N2, or N3 optionally comprises a phosphorothioateinternucleotide linkage.
 9. The composition according to claim 4,wherein the three [N] nucleotides of formula (A) are represented as5′-[N1, N2, N3]-3′, wherein: a. N1 is a 2′-deoxy-2′-fluoro nucleotide,N2 is -deoxy-2′-fluoro nucleotide, and N3 is a 2′-deoxy-2′-fluoronucleotide; and b. any of N1, N2, or N3 optionally comprises aphosphorothioate internucleotide linkage.
 10. The composition accordingto claim 1, wherein X1 is 2 and X2 is
 2. 11. The composition accordingto claim 1, wherein X5 is 3, X1 is 2 and X2 is
 2. 12. The compositionaccording to claim 1, wherein said double-stranded short interferingnucleic acid (siNA) molecule includes one or more universal basesubstitutions.
 13. The composition according to claim 1, wherein saiddouble-stranded short interfering nucleic acid (siNA) molecule includesone or more LNA substitutions.
 14. The composition according to claim 1,wherein the nucleotide at position 14 from the 5′-end of the antisensestrand of said double-stranded short interfering nucleic acid (siNA)molecule is a 2′-deoxy-2′-fluoro nucleotide.
 15. The compositionaccording to claim 1, wherein one or more overhang nucleotides of saiddouble-stranded short interfering nucleic acid (siNA) molecule is a2′-O-methyl nucleotide.
 16. The composition according to claim 1,wherein said double-stranded short interfering nucleic acid (siNA)molecule includes at least one phosphorothioate internucleotide linkage.17. The composition according to claim 1, wherein X5=3; each X1 and X2=1or 2; X3=18, 19, 20, 21, 22, or 24, and X4=17, 18, 19, 20, 21, 22, or23.
 18. The composition according to claim 1, wherein X5=3; each X1 andX2=2; X3=19, and X4=16.
 19. A double-stranded short interfering nucleicacid (s NA) molecule that inhibits the expression of a target gene viaRNA interference, having a sense strand and an antisense strand andcomprising formula (A):

wherein, the upper strand is the sense strand and the lower strand isthe antisense strand of the double-stranded nucleic acid molecule;wherein the antisense strand comprises a sequence having at least 15nucleotides that are complementary to a target RNA sequence encoded bythe target gene and the sense strand comprises a sequence that iscomplementarity to the antisense strand; each N is independently anucleotide which is unmodified or chemically modified or is optionally anon-nucleotide; each B is independently a terminal cap that is presentor absent; (N) represents overhanging nucleotides, each of which isindependently unmodified or chemically modified; [N] representsnucleotides at the 5′-terminus of the antisense strand; X1 and X2 areindependently integers from 0 to 4; X3 is an integer from 15 to 30; X4is an integer from 12 to 27; and X5 is an integer from 1-6, providedthat the sum of X4 and X5 is an integer from 15-30; and wherein (a) allpyrimidine nucleotides in N_(X4) positions are 2′-O-alkyl nucleotides;(b) all purine nucleotides in N_(X4) positions are 2′-halo nucleotides;(c) all pyrimidine nucleotides in N_(X3) positions are 2′-O-alkylnucleotides; (d) all purine nucleotides in N_(X3) positions are 2′-halonucleotides; and (e) [N] position nucleotide(s) are any combination ofribonucleotides, deoxyribonucleotides, 2′-O-alkyl nucleotides, or2′-halo nucleotides; (f) the nucleotide at position 14 from the 5′-endof the antisense strand is a 2′-deoxy-2′-fluoro nucleotide regardless ofwhether it is a purine or pyrimidine; and (g) [N] nucleotides of formula(A) are represented as 5′-[N1, N2, N3]-3′, wherein i) N1 is a 2′-deoxynucleotide, N2 is 2′-deoxy-2′-fluoro nucleotide, and N3 is a 2′-deoxynucleotide; or ii) N1 is a 2′-deoxy-2′-fluoro nucleotide, N2 is2′-deoxy-2′-fluoro nucleotide, and N3 is a 2′-deoxy-2′-fluoronucleotide; or iii) N1 is a 2′-deoxy-2′-fluoro nucleotide, N2 is2′-deoxy-2′-fluoro nucleotide, and N3 is a 2′-deoxynucleotide; or iv) N1is a 2′-deoxy nucleotide, N2 is 2′-deoxy-2′-fluoro nucleotide, and N3 isa 2′-O-methyl nucleotide; or v) N1, N2, and N3 are all ribonucleotideshaving phosphorothioate internucleotide linkages.
 20. A polymercomprising the double-stranded short interfering nucleic acid (siNA)molecule according to claim
 19. 21. A compound comprising thedouble-stranded short interfering nucleic acid (siNA) molecule accordingto claim 19 covalently attached to a ligand.
 22. A lipid nanoparticlecomposition comprising the double-stranded short interfering nucleicacid (siNA) molecule according to claim
 19. 23. The lipid nanoparticlecomposition according to claim 22, further comprising: (a)(13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine; (b)cholesterol; (c) DSPC; and (d) PEG-DMG.
 24. The lipid nanoparticlecomposition according to claim 23, wherein the(13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, cholesterol,DSPC, and PEG-DMG have a molar ratio of 50:30:10:2 respectively
 25. Acomposition comprising the double-stranded short interfering nucleicacid (siNA) molecule according to claim 19 and a pharmaceuticallyacceptable carrier or diluent.